Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

BACKGROUND: The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. STUDY DESIGN AND METHODS: Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. RESULTS: HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. CONCLUSION: ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells.

Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype.

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.

Mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a Bm subgroup individual.

BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.

Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity.

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA-related SNPs were measured. Significant correlations between genotype in each RA-related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.

Genetic variation of FUT2 in a Vietnamese population: identification of two novel Se enzyme-inactivating mutations.

BACKGROUND: The human FUT2 gene encodes a secretor-type α(1,2)fucosyltransferase, and many population-specific polymorphisms have been reported in the coding region. STUDY DESIGN AND METHODS: Direct sequencing, real-time polymerase chain reaction, and high-resolution melt (HRM) analysis were done to detect single-nucleotide polymorphism (SNPs) and copy number variations (CNVs) in a Vietnamese population. The impacts of two novel mutations on the encoded enzyme were examined by a transient expression study. RESULTS: The major nonfunctional allele in the 294 Vietnamese was se(357,385), whereas no CNV was detected. Two novel SNPs, 818C>A (Thr273Asn) and 853G>A (Ala285Thr), distributed at low frequency, were shown to remarkably affect the enzyme activity. CONCLUSION: The allelic polymorphism of FUT2 in Vietnamese is similar to that of other East and Southeast Asian populations. This result may reflect the history and gene flow of this population. In addition, HRM analysis seems to be a simple and effective method for screening rare SNPs of FUT2 in a large number of samples. [Correction statement added after online publication 21-Dec-2011: Thr273Ala has been updated to Thr273Asn throughout.]

Detection of allogeneic blood group A and B enzyme activities in patients with ABO incompatible kidney transplantation.

The phenomenon of accommodation in recipients of blood group ABO incompatible kidney transplantation (iKTx), in which grafts survive despite the presence of blood group A or B antigen in the graft and the presence of corresponding antibodies in the recipient's blood, is not uncommon. alpha1,3-N-Acetylgalactosaminyltransferase and alpha1,3galactosyltransferase associated with the synthesis of blood group A and B antigen (A and B enzymes), respectively, were measured by a highly specific enzyme-linked immunosorbent assay in the sera and transplanted tissues of patients who underwent an ABO iKTx. Allogeneic A and B enzymes were present in the sera and tissues as well as A and B antigens in the tissues for a long period, which hitherto have never been seen in recipients prior to an iKTx. However, activities of these enzymes in the sera after an iKTx decreased in patients who experienced a serious acute antibody-mediated rejection and disappeared in patients who had an unrepairable rejection, leading to graft loss without establishment of accommodation. Our observations on the presence of allogeneic A and B enzymes in the recipients' sera should have implications in decision making for a successful iKTx.

Genetic and expression analysis of all non-synonymous single nucleotide polymorphisms in the human deoxyribonuclease I-like 1 and 2 genes.

Members of the human DNase I family, DNase I-like 1 and 2 (DNases 1L1 and 1L2), with physiological role(s) other than those of DNase I, possess three and one non-synonymous SNPs in the genes, respectively. However, only limited population data are available, and the effect of these SNPs on the catalytic activity of the enzyme remains unknown. Genotyping of all the non-synonymous SNPs was performed in three ethnic groups including six different populations using the PCR-RFLP method newly developed. Asian and African groups including Japanese, Koreans, Ghanaians and Ovambos were typed as a single genotype at each SNP, but polymorphism at only SNP V122I in DNase 1L1 was found in Caucasian groups including Germans and Turks; thus a Caucasian-specific allele was identified. The DNase 1L1 and 1L2 genes show relatively low genetic diversity with regard to these non-synonymous SNPs. The level of activity derived from the V122I, Q170H and D227A substituted DNase 1L1 corresponding to SNPs was similar to that of the wild-type, whereas replacement of the Asp residue at position 197 in the DNase 1L2 protein with Ala, corresponding to SNP D197A, reduced its activity greatly. Thus, SNP V122I in DNase 1L1 exhibiting polymorphism exerts no effect on the catalytic activity, and furthermore SNP D197A in DNase 1L2, affecting its catalytic activity, shows no polymorphism. These findings permit us to postulate that the non-synonymous SNPs identified in the DNase 1L1 and 1L2 genes may exert no influence on the activity levels of DNases 1L1 and 1L2 in human populations.

Comparison of methods of detecting bacterial biofilm.

Fresh bacterial biofilm produced by Staphylococcus epidermidis was sensitively detected by using a chemiluminescent probe. Its lower detection limit was around 10(-5) - 10(-4) dilution (equivalent to 10(-5) - 10(-4) cm2 biofilm) - 10(2) - 10(3) times as sensitive as ordinary colorimetric methods. The linearity and dynamic range were slightly better than those of fluorometry.

11-Tungstophosphate with iron(II) and hydrogen peroxide efficiently detached bacterial biofilm.

An attempt was made to detach bacterial biofilm, formed by Staphylococcus epidermidis, by using hydrogen peroxide (H(2)O(2)) and tungsten compounds. When iron(II) (Fe(2+)) was mixed with undecatungstophosphate ([PW(11)O(39)](7-)) and then H(2)O(2), the resulting mixture was able to totally remove the biofilm probably because of co-generation of (1)O(2) and .OH. A mixture of undecatungstosilicate ([SiW(11)O(39)](8-)) and Fe(2+) (or Cu(2+)) also gave a good result, but their catalytic activities for producing .OH (or (1)O(2)) were rather weak. An electron microscopic study showed that almost nothing was visible on the surface of a biofilm-coated glass after treatment with 1mM [PW(11)O(39)](7-)+1 mM Fe(2+) and 500 mM H(2)O(2) (incubated for 1 h at 37 degrees C).

Genotyping of five single nucleotide polymorphisms in the OCA2 and HERC2 genes associated with blue-brown eye color in the Japanese population.

Human eye color is a polymorphic phenotype influenced by multiple genes. It has recently been reported that three single nucleotide polymorphisms (SNPs) within intron 1 of the OCA2 gene (rs7495174, rs4778241, rs4778138) and two SNPs in intron 86 (rs12913832) and the 3' UTR region (rs1129038) of the HERC2 gene--located in the upstream of the OCA2 locus--have a high statistical association with human eye color. The present study is the first to examine in detail the genotype and haplotype frequencies for these five SNPs in an Asian (Japanese) population (n = 523) comprising solely brown-eyed individuals. Comparison of the genotype and haplotype distributions in Japanese with those in African and European subjects revealed significant differences between Japanese and other populations. Analysis of haplotypes consisting of four SNPs at the HERC2-OCA2 locus (rs12913832/rs7495174/rs4778241/rs4778138) showed that the most frequent haplotype in the Japanese population is A-GAG (0.568), while the frequency of this haplotype is rather low in the European population, even in the brown-eyed group (0.167). The haplotype distribution in the Japanese population was significantly different from that in the brown-eyed European group (F(ST) = 0.18915).

Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1.

We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene (DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.

Extremely high prevalence of DNASE1*1 allele in African populations.

The single nucleotide polymorphism (SNP) at deoxyribonuclease I (DNase I), designated as DNASE1 (NCBI SNP number; 1053874), in exon 8 (A2317G) has been shown to be associated with liver disease, colorectal carcinoma, and gastric carcinoma in Japanese patients. In this study, we investigated the frequency of the DNASE1 polymorphism in Ghanaian (n = 96) and Xhosa (n = 78) populations and compared the results with those of other studies. The single nucleotide polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The frequencies of DNASE1*1 in the Ghanaian and Xhosa populations were 0.90 and 0.88, respectively. These two African populations had an extremely high frequency of DNASE1*1, similar to that of the Ovambos living in Namibia. Caucasians and Asians had a lower frequency of DNASE1*1 than the African groups. This study is the first to reveal an extremely high frequency of DNASE1*1 among African populations.

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I.

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation.

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

A case of sudden unexpected infant death involving a homozygotic twin with the thermolabile CPT2 variant, accompanied by rotavirus infection and treatment with an antibiotic containing pivalic acid.

We investigated a case of sudden unexpected death involving a 22-month-old male homozygotic twin infant. After both of the twins had suffered from gastroenteritis, one was found dead in his bed, but his brother survived and has since been healthy. Notably, only the deceased had been treated with an antibiotic containing pivalic acid, which may sometimes cause hypocarnitinemia. Postmortem computed tomography and medicolegal autopsy demonstrated severe liver steatosis, and subsequent genetic analysis revealed that the twin had the thermolabile variant of carnitine palmitoyl transferase 2 (CPT2). On the basis of these facts, we concluded that the cause of death had been fatty acid oxidation deficiency accelerated by an antibiotic containing pivalic acid and virus infection in this infant harboring the thermolabile genetic variant of CPT2. Although each factor alone was not fatal, their combination appeared to have resulted in sudden unexpected infant death.

Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine.

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

Brain fragility can be estimated by its putrefactive signs on postmortem computed tomography.

Along with time after death, postmortem computed tomography (PMCT) of the brain can reveal sequential changes. In the present study, we investigated the relationship between brain rigidity and advanced postmortem changes such as intravascular gas production, cerebral settling or cerebral liquefaction on PMCT. We then examined the findings of PMCT as an indicator of successful macroscopic examination of arbitrary brain slices at classical autopsy. The association between these advanced postmortem changes and the validity of macroscopic brain examination was investigated in 149 cases that were examined by PMCT at our department prior to autopsy in the period from September 2011 to December 2013. We found that the postmortem changes, classified into four stages, generally reflected the fragility of the brain. Thus, it is likely that PMCT findings of advanced postmortem changes are able to indicate decreased brain rigidity ahead of autopsy. These findings support the idea that PMCT could be used as a guide by forensic pathologists for suitable handling of a fragile brain, thus enhancing the quality of autopsy.

Abrupt pubertal elevation of DNase I gene expression in human pituitary glands of both sexes.

Deoxyribonuclease I (DNase I) was confirmed to be expressed in the human pituitary gland, particularly the anterior lobe, at levels comparable to those in the pancreas. The DNase I activity and the amount of gene transcript present in the pituitary glands of individuals aged from 1 month to 89 years was significantly age-dependent, with an abrupt elevation after the neonatal and prepubertal periods irrespective of gender, followed by a gradual age-dependent decline in males and a marked reduction in females in their postreproductive period. This DNase I age dependence in the pituitary gland was not present in the pancreas and serum. These observations suggest that tissue-specific up-regulation of DNase I gene expression in the pituitary gland occur, possibly at the onset of puberty.

Molecular, biochemical and immunological analyses of canine pancreatic DNase I.

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.