Sequence analysis of ABO and its homologues is valid for species identification.

BACKGROUND: ABO and its paralogues, such as A3GALT2 and GGTA1, encoding α1,3-Gal(NAc) transferases, belong to the glycosyltransferase 6 (GT6) gene family. We have developed an alternative method for the identification of species based on sequence variations within the GT6 gene family, which is applicable to degraded DNA. METHODS/MATERIALS: DNA samples prepared from control mammalian species, together with an unknown sample, were polymerase chain reaction (PCR)-amplified using one universal primer pair targeting the sequences in the last coding exons of the GT6 gene family, yielding 141-bp products derived from those multiple loci. After cloning, sequence determination and Basic Local Alignment Search Tool analysis, phylogenetic trees were constructed. RESULTS: Comparison of the sequences obtained with those references showed good concordance with each of the starting species of mammals. This system was able to identify 'mouse' or 'rodent' as the origin of the unknown sample. CONCLUSION: For the identification of species, genotyping of ABO and its homologues would be applicable for the analysis of degraded DNA samples. Although the method employed in this study is likely valid for mammals, it would not be suitable for birds, fish and reptiles. It may be possible to improve the present method for use with other species by employing an alternative universal primer set.

Presence of nucleotide substitutions in the ABO promoter in individuals with phenotypes A3 and B3.

Recently, the involvement of mutation and deletion of transcription regulatory elements in the Bm , Am , A3 and B3 phenotypes has been reported. In the present study, we carried out genetic analysis of individuals with A3 and B3 using peptide nucleic acid-clamping PCR to exclude amplification of O alleles. Two single-point mutations, -76G>C and -68G>T, were found in the ABO promoter on the A-allele in three A3 individuals and on the B allele in a B3 individual, respectively. Transient transfection of luciferase reporter plasmids carrying the same mutations into K562 cells revealed decreased luciferase activity in comparison with that carrying the wild-type promoter. These observations suggest that the mutations downregulate the promoter activity, leading to reduction in A- or B-antigen expression on red blood cells in individuals with the A3 and B3 phenotypes.

ABO alleles are linked with haplotypes of an erythroid cell-specific regulatory element in intron 1 with a few exceptions attributable to genetic recombination.

Recent investigation of transcriptional regulation of the ABO genes has identified a candidate erythroid cell-specific regulatory element, named the +5·8-kb site, in the first intron of ABO. Six haplotypes of the site have been reported previously. The present genetic population study demonstrated that each haplotype was mostly linked with specific ABO alleles with a few exceptions, possibly as a result of hybrid formation between common ABO alleles. Thus, investigation of these haplotypes could provide a clue to further elucidation of ABO alleles.

A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently.

Blood group B gene is barely expressed in in vitro erythroid culture of Bm-derived CD34+ cells without an erythroid cell-specific regulatory element.

BACKGROUND AND OBJECTIVES: Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals. MATERIALS AND METHODS: We carried out in vitro erythroid differentiation of CD34(+) cells from peripheral blood of a Bm individual harbouring a 3.0-kb deletion including an erythroid cell-specific regulatory element, named the +5.8-kb site, in intron 1 of the human ABO blood group gene. RESULTS: During the in vitro differentiation of CD34(+) cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5.8-kb site in cultured erythroid cells expressing ABO. CONCLUSION: It is likely that the +5.8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1.

Presence of nucleotide substitutions in transcriptional regulatory elements such as the erythroid cell-specific enhancer-like element and the ABO promoter in individuals with phenotypes A3 and B3, respectively.

BACKGROUND AND OBJECTIVES: An erythroid cell-specific regulatory element, referred to as the +5.8-kb site, has been identified in the first intron of the human ABO blood group gene. Subsequent studies have revealed involvement of deletion or mutation at the site in phenotypes Am , Bm and ABm . We investigated the molecular mechanisms involved in the A3 and B3 phenotypes. MATERIALS AND METHODS: Genomic DNAs were prepared from peripheral blood of seven A3 individuals and twelve B3 or AB3 individuals, and the nucleotide sequences were investigated using PCR and sequencing. Promoter assays were performed with K562 cells. RESULTS: Two single point-mutations at +5893 or +5909 in the site on the A-allele were found in A3 individuals, while promoter assays revealed decreased activity at the site as a result of each substitution. In two B3 individuals, a single point-mutation at -77 in the ABO promoter on the B-allele was found, and the substitution was demonstrated to reduce the promoter activity. CONCLUSION: Nucleotide substitutions in the transcriptional regulatory elements such as the +5.8-kb site and the ABO promoter appear to decrease transcription from the A- and B-alleles, resulting in reduction in A- and B-antigen expression in A3 and B3, respectively.

Deletion of the RUNX1 binding site in the erythroid cell-specific regulatory element of the ABO gene in two individuals with the Am phenotype.

BACKGROUND AND OBJECTIVES: An erythroid cell-specific regulatory element, referred to as the +5·8-kb site, had been identified in the first intron of the human ABO blood group gene. Subsequent studies revealed that either a 5·8-kb deletion including the +5·8-kb site or disruption of a GATA factor binding motif at the site was present in all Bm and ABm individuals examined. We investigated the molecular mechanism of the Am phenotype, which is analogous to the Bm phenotype. MATERIALS AND METHODS: Genomic DNAs were prepared from peripheral blood of two Am individuals, and the nucleotide sequences were investigated using PCR and direct sequencing. Electrophoretic mobility shift assay (EMSA) and promoter assay with K562 cells were carried out. RESULTS: A novel 23-bp nucleotide deletion was found at the +5·8-kb site in both individuals. EMSAs demonstrated binding of the transcription factor RUNX1 to the nucleotides within the deletion. Promoter assays showed that the deletion reduced the transcriptional activity of the +5·8-kb site. CONCLUSION: Deletion of the 23-bp nucleotides including the RUNX1 binding site decreases transcription of the A allele, resulting in the reduction in A antigen expression in the Am phenotype.

A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family.

We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5-7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.

Characterization of the human ABO gene promoter in erythroid cell lineage.

BACKGROUND: The human ABO blood group system is important in transfusion and organ transplantation. Although the molecular basis of the ABO gene has been established, recent studies have begun to characterize the mechanism of the ABO gene expression. MATERIALS AND METHODS: Transient transfection assays were carried out in human erythroleukaemia HEL cells and human gastric cancer KATOIII cells. Electrophoretic mobility shift assays were performed using nuclear extracts derived from both cells. RESULTS: Our characterization of the 5'-upstream sequence of the ABO genes indicated that the region between -117 and +31 is essential to direct expression of a reporter gene in erythroid cells. We show that a sequence located between positions -22 and -14 of the ABO promoter binds a ubiquitous transcription factor Sp1 or Sp1-like protein(s). Mutation of this site abrogates binding of those factors and reduces the ability of the ABO promoter to function in erythroleukaemia cells and gastric cancer cells. CONCLUSION: The expression of the ABO promoter appears to be influenced by the binding of Sp1 or Sp1-like protein(s) in both erythroid and epithelial cell lineages.

Garlic supplementation increases testicular testosterone and decreases plasma corticosterone in rats fed a high protein diet.

The effects of garlic supplementation on protein metabolism were investigated by measuring testis testosterone and plasma corticosterone in rats fed diets with different protein levels. In Experiment 1, rats were fed experimental diets with different protein levels (40, 25 or 10 g/100 g casein) with or without 0.8 g/100 g garlic powder. After 28 d of feeding, testosterone contents in the testis were significantly higher and plasma corticosterone concentrations were significantly lower in rats fed 40 and 25% casein diets with garlic powder than in those fed the same diets without garlic powder. Urinary excretion of 17-ketosteroid (an index of testosterone), nitrogen balance and hepatic arginase activity were significantly higher in rats fed the 40% casein diet with garlic powder than in the 40% casein controls. In Experiment 2, the effect of diallyldisulfide (a major volatile sulfur-containing compound in garlic) on the secretion of luteinizing hormone (LH) from the pituitary gland, which regulates testosterone production in the testis, was investigated in anesthetized rats. Plasma LH concentration increased dose dependently after administration of diallyldisulfide (P < 0.01, r = 0.558). These results suggest that dietary supplementation with 0.8 g/100 g garlic alters hormones associated with protein anabolism by increasing testicular testosterone and decreasing plasma corticosterone in rats fed a high protein diet.

Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodes a glycosyltransferase with both A and B transferase activity.

We have cloned murine genomic and complementary DNA that is equivalent to the human ABO gene. The murine gene consists of at least six coding exons and spans at least 11 kilobase pairs. Exon-intron boundaries are similar to those of the human gene. Unlike human A and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an enzyme with both A and B transferase activities, and this cis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishing a mouse model system to assess the functionality of the ABO genes in the future.

Regional differences in homicide patterns in five areas of Japan.

This article describes regional differences in the homicide patterns which occurred in Sapporo City and the surrounding area, and in Akita, Ibaraki, Chiba and Toyama prefectures in Japan. Information collected from each case of homicide included factors such as age, sex of the victim and assailant, causes of death, disposition of the offender, relationship between assailant and victim, reasons for criminal action, et al. The statistical features of homicidal episodes among the five different regions showed considerable variation, as follows. The mean death rates for homicide (number of victims per 100,000 of population) during the period 1986-1995 were 0.44 (Sapporo), 0.8 (Akita), 0.58 (Toyama), 0.7 (Ibaraki) and 0.75 (Chiba), respectively. Close family relationship between the victim and assailant was observed in the homicidal acts which occurred in Sapporo, Akita and Toyama. Assailant's relationship to victim was commonly extra-familial in Ibaraki and Chiba-neighboring megalopolis Tokyo, where some events of murder by a foreigner occurred. Homicide by female assailant, murder by mentally abnormal killers and homicide-suicide events were closely associated with family members. And these factors contributed to the considerable number of victims in Sapporo, Akita and Toyama. But, this close family relationship of the victim to the assailant did not correspond with the elevation in the number of deaths, and it was rather inversely related to the higher death rates recognized in Ibaraki and Chiba. This comparative study suggested that rapid urbanization considerably affects regional differences in homicide patterns.

Acute cerebellar hemorrhage in a patient with Klinefelter syndrome: XXY karyotype obtained postmortem from cells from pericardial fluid.

A case of Klinefelter syndrome and a spontaneous cerebellar hemorrhage in a 12-year-old boy is presented. Autopsy revealed that the hemorrhage was due to the rupture of a dilated artery in an arteriovenous malformation in the right cerebellar hemisphere. The small, undescended testes exhibited partial atrophy of the seminiferous tubules. Postmortem chromosome analysis of cells from the pericardial fluid demonstrated a 47, XXY karyotype. He had previous surgical treatment for bilateral thumb polydactyly and patent ductus arteriosus. In juvenile cases of sudden death with overlapping morphological dysgenesis, postmortem karyotyping may provide important diagnostic information.

Progress in the study of ABO blood group system.

Progress in the study of ABO blood group system during the last three decades was reviewed according to following 5 items. 1. Structure of H-, A- and B-active saccharides isolated from the globoside fractions from human erythrocytes. 2. Enzyme characterization of a blood group A-gene specified alpha-N-acetyl-galactosaminyltransferase (A-enzyme), and a blood group B-gene specified alpha-galactosyltransferase (B-enzyme). 3. Immunological properties of the A- and B-enzyme. 4. The cDNA structures of human blood group ABO genes. 5. Transcriptional regulation of the human blood group ABO genes.

Expression of human histo-blood group ABO genes is dependent upon DNA methylation of the promoter region.

We have investigated the regulatory role of DNA methylation in the expression of the human histo-blood group ABO genes. The ABO gene promoter region contains a CpG island whose methylation status correlates well with gene expression in the cell lines tested. The CpG island was found hypomethylated in some cell lines that expressed ABO genes, whereas the other cell lines that did not express ABO genes were hypermethylated. Whereas constitutive transcriptional activity of the ABO gene promoter was demonstrated in both expressor and nonexpressor cell lines by transient transfection of reporter constructs containing the ABO gene promoter sequence, HhaI methylase-catalyzed in vitro methylation of the promoter region prior to DNA transfection suppressed the promoter activity when introduced into the expressor gastric cancer cell line KATOIII cells. On the other hand, in the nonexpressor gastric cancer cell line MKN28 cells, treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation of the ABO gene promoter and appearance of A-transferase messages, as well as A-antigens synthesized by A-transferase. Taken together, these studies suggest that DNA methylation of the ABO gene promoter may play an important role in the regulation of ABO gene expression.

Phage display cDNA cloning of protein with carbohydrate affinity.

Cell surface complex carbohydrate structures that are synthesized through the actions of glycosyltransferases play an important role in cell-to-cell and cell-to-extracellular matrix interactions. To examine the feasibility of phage display technique to clone cDNAs encoding glycosyltransferases, we performed biopanning experiments using human histo-blood group A transferase as a model enzyme and its substrate, blood group H-specific glycoproteins, as a bait ligand. Our attempts have been unsuccessful, possibly because of the enzyme's weak affinity with the target. However, we have selectively enriched several phage clones that expressed capsid proteins fused with galectin-3, a galactose/lactose-specific animal lectin of the galectin family. These results demonstrate that this novel approach of phage display is useful in cDNA cloning of proteins with carbohydrate-binding property.

Allyl-containing sulfides in garlic increase uncoupling protein content in brown adipose tissue, and noradrenaline and adrenaline secretion in rats.

The effects of garlic supplementation on triglyceride metabolism were investigated by measurements of the degree of thermogenesis in interscapular brown adipose tissue (IBAT), and noradrenaline and adrenaline secretion in rats fed two types of dietary fat. In Experiment 1, rats were given isoenergetic high-fat diets containing either shortening or lard with or without garlic powder supplementation (8 g/kg of diet). After 28 d feeding, body weight, plasma triglyceride levels and the weights of perirenal adipose tissue and epididymal fat pad were significantly lower in rats fed diets supplemented with garlic powder than in those fed diets without garlic powder. The content of mitochondrial protein and uncoupling protein (UCP) in IBAT, and urinary noradrenaline and adrenaline excretion were significantly greater in rats fed a lard diet with garlic powder than in those fed the same diet without garlic. Other than adrenaline secretion, differences due to garlic were significant in rats fed shortening, also. In Experiment 2, the effects of various allyl-containing sulfides present in garlic on noradrenaline and adrenaline secretion were evaluated. Administration of diallyldisulfide, diallyltrisulfide and alliin, organosulfur compounds present in garlic, significantly increased plasma noradrenaline and adrenaline concentrations, whereas the administration of disulfides without allyl residues, diallylmonosulfide and S-allyl-L-cysteine did not increase adrenaline secretion. These results suggest that in rats, allyl-containing sulfides in garlic enhance thermogenesis by increasing UCP content in IBAT, and noradrenaline and adrenaline secretion.

DNA polymorphisms in the 5'-flanking sequence of human ABO blood group genes and their association with the alleles for the common ABO phenotypes.

Previously we found that minisatellite located on the 5'-flanking sequence far from the transcription start site of the human ABO blood group genes and containing four tandem repeats of a 43 base pair (bp) consensus sequence was an enhancer element for the transcription of the ABO genes. Concerning the number of tandem repeats in the minisatellite, there are at least three alleles: ABOU1*1, ABOU1*3, and ABOU1*4. Besides the variability of the minisatellite, another location of the 5'-flanking sequence has two alleles associated with the insertion of the 35 bp DNA segment: ABOU2*0 allele with an uneventful sequence and ABOU2*1 allele with the 35-bp insertion, respectively. Both of the DNA polymorphisms are closely associated with the common alleles of blood group ABO phenotypes. Namely, the 5'-flanking configuration consisting of the alleles ABOU1*1 and ABOU2*0 links up with the common A1 allele, whereas, the other 5'-flanking configuration consisting of the alleles ABOU1*4 and ABOU2*1 links up with common B and O alleles (abbreviated as 1-0-A1, 4-1-B and 4-1-O). In these linkages between three loci of alleles of 102 unrelated healthy individuals, two are not consistent with the rules described above. One is 3-1-O and the other is 4-1-A2. These findings suggest that the 3 or 4 times repeats of a 43 bp consensus sequence at ABOU1 locus and the 35 bp-insertion at ABOU2 locus found in 5'-flanking sequence of the B and O genes are incidental to the ABO gene evolution, subject to the ancestral A1 gene.

Transcription of human ABO histo-blood group genes is dependent upon binding of transcription factor CBF/NF-Y to minisatellite sequence.

We have studied the transcriptional regulatory mechanism of the human histo-blood group ABO genes, and identified DNA cis-elements and trans-activating protein that control the expression of these genes which are important in blood transfusion and organ transplantation. We introduced the 5'-upstream sequence of ABO genes into the promoterless reporter vector and characterized the promoter activity of deletion constructs using transient transfection assays with gastric cancer cell line KATO III cells. The sequence just upstream of the transcription start site (cap site), and an enhancer element, which is located further upstream (between -3899 and -3618 base pairs (bp) from the transcription initiation site) and contains 4 tandem copies of a 43-bp repeat unit, were shown in gastric cancer cells to be responsible for the transcriptional activity of the ABO genes. DNA binding studies have demonstrated that a transcription factor, CBF/NF-Y, bound to the 43-bp repeat unit in the minisatellite. Functional importance of these CBF/NF-Y-binding sites in enhancer activity was confirmed by transfection experiments using reporter plasmids with mutated binding sites. Thus, transcriptional regulation of the human ABO genes is dependent upon binding of CBF/NF-Y to the minisatellite.

Human T-cell leukemia virus type I Tax transactivates the promoter of human prointerleukin-1beta gene through association with two transcription factors, nuclear factor-interleukin-6 and Spi-1.

The human T-cell leukemia virus type I (HTLV-I), which infects a wide variety of mammalian cells including monocytes and macrophages, encodes a transactivating protein designated as Tax. We now report that Tax induces the human prointerleukin-1beta (IL1B) gene promoter in monocytic cells. In our transient transfection assays using human THP-1 monocytic cells, a chloramphenicol acetyltransferase (CAT) construct containing the IL1B promoter sequence between positions -131 and +12 showed an approximately 90-fold increase in activity following cotransfection of a Tax expression vector. Moreover, Tax synergized with lipopolysaccharide (LPS) to induce the IL1B promoter activity. Analyses of specific nucleotide substitutions further indicated that the Tax-induced transcriptional activation requires two transcription factor binding motifs within the IL1B promoter; one is a binding site for nuclear factor (NF)-IL6 (CCAAT/enhancer binding protein beta, C/EBP beta), which belongs to the basic region-leucine zipper (bZIP) family and the other for Spi-1 (PU.1), which is an Ets family protein found principally in monocytes, macrophages, and B lymphocytes. In electrophoretic mobility shift assays (EMSA) using in vivo THP-1 nuclear extracts, Tax expression in THP-1 monocytic cells significantly increased binding of the two factors to their target IL1B promoter sequences. However, in contrast to NF-IL6 and Spi-1, DNA binding activity of Oct-1, an ubiquitously expressed octamer-binding protein was not affected by Tax. Additional EMSA using in vitro translated proteins also showed that recombinant Tax enhances DNA binding of both of recombinant NF-IL6 and Spi-1 proteins. These data were supported by our glutathione S-transferase (GST)-pull-down data, which indicated that Tax physically interacts with the two proteins. Based on the results obtained from the present study, we conclude that the IL1B promoter is a Tax-responsive sequence as a result of ability of Tax to induce binding of NF-IL6 and Spi-1 to the IL1B promoter sequence through protein-protein interaction.