Kouichi Mogi

A Simple Method of DNA Extraction and STR Typing from Urine Samples Using a Commercially Available DNA/RNA Extraction Kit*

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.

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

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.

The molecular basis for genetic polymorphism of human deoxyribonuclease II (DNase II): a single nucleotide substitution in the promoter region of human DNase II changes the promoter activity.

Deoxyribonuclease II (DNase II) levels in human vary depending on whether the individual has the DNASE2*H (high) allele or the DNASE2*L (low) allele. We examined the promoter activity of the 5′‐flanking region of each of these alleles by transient transfection luciferase assay. DNASE2*H had 5‐fold higher promoter activity than DNASE2*L in human hepatoma HepG2 cell. Comparison of the nucleotide sequences of the proximal promoter regions revealed a G to A transition at position −75; G and A residues were assigned to DNASE2*H and *L, respectively. Since no differences were found between the open reading frame sequences of these alleles, it is likely that the A−75G transition causes the allelic difference in the promoter activity of the gene, underlying the genetic polymorphism.

Geographical North-South Decline in DNASE1*2 in Japanese Populations

Allele frequencies for human deoxyribonuclease I (DNase I) phenotypes were determined using blood samples from about 2000 Japanese subjects living in nine prefectures, and compared with one another. DNase I phenotyping was performed principally using isoelectric focusing electrophoresis and activity staining. The DNase I system was shown to have enhanced potential for anthropologic, genetic, and clinical studies of Japanese populations. DNase I phenotypes were analyzed to evaluate the degree of genetic variation at the DNASE1 locus. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1allele.

Use of Human Recombinant DNase I Expressed in COS-7 Cells as an Immunogen to Produce a Specific Anti-DNase I Antibody

To obtain human deoxyribonuclease I (DNase I) as an immunogen, we have developed a procedure that is more useful and effective than the conventional procedure, which uses human urine as a starting material. In the new procedure, we culture COS-7 cells transfected with expression vector carrying human DNase I cDNA, and then purify the enzyme from the culture medium. The enzyme can be easily isolated to apparent homogeneity by passage through only three chromatography columns. The rabbit antiserum that we used against the recombinant DNase I was not inferior to that used against DNase I from human urine, in terms of both its ability to discriminate DNase I phenotypes and its ability to neutralize enzyme activity. Therefore, our procedure may be useful for producing an antibody specific for human DNase I.

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.

Postmortem absorption of dichloromethane: a case study and animal experiments.

Abstract A case of accidental death after occupational exposure to an atmosphere containing dichloromethane (DCM) is reported. The concentrations of DCM in the blood and tissues of a 40-year-old man who died while observing an industrial washing machine filled with DCM vapour were blood 1660 mg/l, urine 247 mg/l, brain 87 mg/ kg, heart muscle 199 mg/kg and lungs 103 mg/kg which are 3–7 times higher than previously reported fatal levels. The body was left undiscovered in the machine filled with DCM vapour for about 20 h. The present study was designed to determine whether all the DCM detected in the tissues and body fluids had been inhaled while alive using rats as the experimental model. The concentrations of DCM in the tissues and body fluids of a rat that died from DCM poisoning and was left for 20 h in a box containing DCM vapour were the same as those in the tissues and body fluids of a rat that had died from an injected overdose of barbiturates and had then been placed in the DCM box in a similar manner. Moreover, the concentrations of DCM in the tissues and body fluids of the carcasses that were exposed to the DCM vapour increased gradually throughout the period of exposure. These findings imply that DCM is able to penetrate the tissues and body fluids of rat carcasses through a route other than inhalation such as through the skin.

Rapid Purification of Human DNase I Using Mouse Monoclonal Anti-DNase I Antibodies and Characterization of the Antibodies

Five anti-human deoxyribonuclease I (DNase I) monoclonal antibodies were obtained from BALB/c mice immunized with DNase I purified from human urine. Four of them inhibited DNase I enzyme activity, as did a rabbit polyclonal antibody; these 4 did not have immunostaining ability. The remaining one had immunostaining ability but no inhibitory activity. A Sepharose 4B column conjugated with 1 of the 4 antibodies that had inhibitory activity effectively adsorbed and eluted the DNase I enzyme; this did not occur with the rabbit polyclonal antibody. We showed that adding an immunoaffinity chromatography step made the purification of human DNase I easier and faster than the conventional procedure.

Molecular Cloning of cDNA Encoding Xenopus laevis Deoxyribonuclease I

A 1200-bp cDNA encoding Xenopus laevis deoxyribonuclease I (X. laevis DNase I) was constructed from the total RNA of a X. laevis pancreas using a rapid amplification of cDNA ends method. When the cDNA was transiently transfected into COS-7 cells, the recom-binant polypeptide exhibited similar enzymological properties to those of the native pancreatic DNase I. The recombinant enzyme was considerably more labile than most other vertebrate DNase I enzymes. The X. laevis DNase I polypeptide was larger than any other known vertebrate DNase I, containing a unique Cys-rich stretch of 68 or 70 amino acid residues at the carboxyl terminus, and it had less well conserved binding sites for the Ca2+, G-actin and DNA, and two DNase I signature motifs. These alterations might account for its heat instability.

A death resulting from inadvertent intravenous infusion of enteral feed

Abstract A female patient suffering from the after-effects of an intracerebral hemorrhage, inadvertently received approximately 50 ml of enteral feed containing high molecular weight dextrin intravenously and died 6 h later despite intensive emergency resuscitation attempts. The total quantity of enteral feed received was calculated from the amounts of dextrin measured in the blood. This is the first report describing how the total quantity of enteral feed administered intravenously was determined using biochemical analysis.