Hidekazu Horiuchi

Association of α2-HS glycoprotein (AHSG, fetuin-A) polymorphism with AHSG and phosphate serum levels

Alpha2-HS glycoprotein (AHSG), also known as fetuin-A, is a plasma protein displaying high-affinity interaction with calcium phosphate, by which ectopic vascular calcification is prevented. This investigation has attempted to evaluate the relationship between AHSG polymorphism and serum levels of AHSG and calcium-related parameters. AHSG levels in unrelated individuals were measured by quantitative rocket immunoelectrophoresis and were 581±38, 542±31, and 494±23mg/l for three major genotypes of AHSG1 homozygotes (n=99), heterozygotes (n=55), and AHSG2 homozygotes (n=22), respectively (differences were significant: P<0.001). The circulating AHSG level was therefore influenced by the genetic polymorphism with the additive reduction in the AHSG2 allele. Statistical analysis of simple and multiple regression models revealed no associations between AHSG levels and serum values of total calcium, albumin-corrected total calcium, and ionized calcium. However, the AHSG levels demonstrated a significant negative correlation with free phosphate levels (P<0.001), indicating that AHSG is a novel determinant of serum phosphate. The AHSG polymorphism is attributable to the hereditary variation of AHSG and phosphate serum levels, which may affect skeletal development and chronic disorders such as vascular calcification.

Expression of the prolactin-inducible protein (PIP/GCDFP15) gene in benign epithelium and adenocarcinoma of the prostate

Prolactin‐inducible protein (PIP), also known as gross cystic disease fluid protein 15, is a predominant secretory protein in various body fluids, including saliva, milk and seminal plasma. Immunohistochemistry of this protein has been exploited as a clinical marker for breast cancer and Pagets disease. This study comparatively examined PIP expression in normal prostate tissues and in adenocarcinomas of the prostate. Quantitative real‐time RT‐PCR revealed low‐level presence (6%) of PIP mRNA in normal prostate tissue in comparison with the seminal vesicle. Indirect immunostaining with monoclonal antibody 3E7 displayed a positive sign for benign epithelium in 8 cases (29.6%) among 27 normal specimens; however, the incidence significantly increased to 56.1% (37/66) in instances involving primary prostate carcinoma tissues of different types. Quantitative RT‐PCR also demonstrated that PIP transcript levels in carcinoma regions were significantly higher than corresponding levels in benign regions. These findings conclusively showed that benign prostate epithelium expresses PIP at low levels; in contrast, PIP is over‐expressed in carcinomas of the prostate.

Sensitive Detection of Polyalanine Expansions in PHOX2B by Polymerase Chain Reaction Using Bisulfite-Converted DNA

Congenital central hypoventilation syndrome, also known as Ondines curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.

Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem repeat in humans.

The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht-Lundborg type. We undertook a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the array should be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate evolution. The variability conceivably developed after the separation to humans.

A death in a stationary vehicle whilst idling : unusual carbon monoxide poisoning by exhaust gases

In this paper, we describe an autopsy case in which death was due to accidental carbon monoxide poisoning occurring in a stationary vehicle idling in an open space. To investigate the source of the fatal fumes, the death scene situation was reconstructed using the vehicle. Exhaust gases were found to invade the interior through the floor from a defective exhaust system. CO gas was detected while idling and the level in the cabin gradually rose to 1.5% over a 2-h period. Since the 8-year-old motor vehicle seemed to have been defective for some months, it was concluded that stationary idling overnight caused an accumulation of toxic gases in the interior.

Molecular characterization of a human monoclonal antibody to B antigen in ABO blood type

A human anti-B antibody of clone BT97 was obtained from a healthy individual of type A of the ABO blood group without immunization. Cloning was performed by means of heterohybridoma formation of cell fusion between human peripheral lymphocytes and mouse myeloma cells. The antibody selectively reacted with B-antigen in flow cytometry using red blood cells and enzyme-linked immunosorbent assay. The VH and VL genes of BT97 were derived from the germline genes of DP-47 and 3p.81A4, respectively, with a couple of somatic mutational events. Comparative analysis with other reported anti-A, B and H antibodies revealed that the amino acid sequence of the VH region was more homologous than that of the VL region. The sequence of BT97 showed complete identity with one anti-H natural antibody reported by Marks et al., with the exception of the CDR3 region. It is not known whether the homologies include the common properties of the natural antibodies; however, a particular germline gene potentially changes to anti-ABH antibodies. We think that this method is suitable for cDNA preparation of human monoclonal antibodies to blood group antigens and for sequence analysis.