Shinjiro Mori

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.

Identification of the three non‐identical subunits constituting human deoxyribonuclease II

We purified DNase II from human liver to apparent homogeneity. The N‐terminal amino acid sequences of each of three components constituting the purified mature enzyme were then separately determined by automatic Edman degradation. A combination of this chemical information and the previously reported nucleotide sequence of the cDNA encoding human DNase II [Yasuda et al. (1998) J. Biol. Chem. 273, 2610–2626] allowed detailed elucidation of the enzymes subunit structure: human DNase II was composed of three non‐identical subunits, a propeptide, proprotein and mature protein, following a signal peptide. Expression analysis of a series of deletion mutants derived from the cDNA of DNase II in COS‐7 cells suggested that although a single large precursor protein may not be necessary for proteolytic maturation, the propeptide region L17–Q46 may play an essential role in generating the active form of the enzyme.

Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I.

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.

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.

Structure and organization of the human deoxyribonuclease II (DNase II) gene

The structure of the human gene for deoxyribonuclease II (DNase II; EC 3.1.22.1) was determined using several specific primers based on the human DNase II cDNA sequence [Yasuda et al. (1998). J. Biol. Chem.273, 2610–2616] in a polymerase chain reaction‐based strategy. The gene spanned about 6 kb and consisted of 6 exons. No canonical TATA or CAAT boxes could be identified within the 1341 nucleotides upstream of the putative transcription start site, although the 5′‐flanking region contained a CpG island and several putative binding motifs for transcription factors Sp1 and ETF. These properties indicate that the DNase II gene is a housekeeping gene and this is compatible with its ubiquitous expression in human tissues. Three different cleavage/polyadenylation sites were identified in the 3′‐flanking region, leading to the production of multiple DNase II mRNA species. However, a comparison of the entire translated sequences of the gene from a pair of subjects with homozygous DNase II phenotypes H and L revealed no differences in the nucleotide sequences.

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.

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.