Yoshihiko Kawarada

Antibody against single-stranded DNA detects both programmed cell death and drug-induced apoptosis

Cyclophosphamide induced fragmented nuclei in mouse thymic epithelial cells. Agarose gel electrophoresis showed the fragmentation of the DNA extracted from mouse thymus exposed to cyclophosphamide. The cell death induced by cyclophosphamide was considered to be apoptotic. Polyclonal antibody against single-stranded DNA was used immunohistochemically to detect apoptotic cell death in thymic epithelial cells. This antibody also detected programmed cell death in the interdigital necrotic zone of the mouse limb plate on day 14 of gestation, and in the ganglion of the trigeminal nerve on day 13 of gestation. These results show that the antibody specific for single-stranded DNA detected both drug-induced apoptosis and programmed cell death during embryogenesis.

Detection of apoptotic cells in human colorectal cancer by two different in situ methods: antibody against single-stranded DNA and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) methods.

Wecomparatively investigated the extent of apoptotic cell loss in human colorectal cancers evaluated by two methods, namely the conventional terminal deoxynucleotidyl transferase (TdT)‐ mediated deoxyuridine triphosphate (dUTP)‐biotin nick end‐labeling (TUNEL) method and immunohistochemistry for single‐stranded (ss) DNA. The apoptotic index (AI) obtained with the TUNEL method was higher than that shown by the immunohistochemistry for ssDNA. However, a significant correlation in AIs evaluated by these methods was found. The AIs obtained by both methods were significantly higher in the advanced cancers than in the early cancers. Cellular proliferation activity was assessed in terms of positivity rate (PR) for expression of proliferating cell nuclear antigen (PCNA). The PR of advanced cancers was significantly higher than that of early cancers. The present results indicate that immunohistochemistry for ssDNA is useful (as is the TUNEL method) for evaluation of apoptotic tumor cells in colorectal carcinomas. In addition, it was confirmed that there is a remarkable increase of not only proliferation activity, but also tumor cell apoptosis in the process of progression of colon cancer from early to advanced stages of the disease.

Copper incorporation into ceruloplasmin in rat livers

Ceruloplasmin, a blue copper oxidase found in plasma, is synthesized in hepatocytes as a single polypeptide chain consisting of a 19 amino acid leader peptide plus 1046 amino acids of mature protein (132 kDa). Holoceruloplasmin is secreted into the plasma with 6-7 atoms of copper bound per molecule. In this study we identified apo- and holoceruloplasmin and examined the mechanism of copper incorporation during ceruloplasmin biosynthesis using the Long-Evans Cinnamon (LEC) rat which does not incorporate copper into newly synthesized ceruloplasmin. We followed the conversion from ceruloplasmin precursor (with little or no carbohydrate) to the larger product (after carbohydrate addition), which occurred in the secretory compartments of hepatocytes, by native gel electrophoresis. We found that copper accumulates in the hepatocellular Golgi apparatus of LEC rats due to a disorder in the process of copper incorporation. The data indicate that copper is incorporated into ceruloplasmin late in the course of its transport through the secretory compartments.

Urinary copper excretion in type 2 diabetic patients with nephropathy.

Background/Aims: The aim of this study was to examine the relationship between the degree of urinary copper excretion and stages of diabetic nephropathy. Methods: Copper, ceruloplasmin and albumin concentrations were measured in serum and urine samples from 41 type 2 diabetic outpatients with different stages of nephropathy and from 10 healthy controls. The copper/albumin and copper/ceruloplasmin ratios in serum and urine were determined. Furthermore, we examined whether free copper ions are dissociated from ceruloplasmin under various pH conditions. Results: Urinary copper concentrations significantly increased only in macroalbuminuric patients. The copper/ceruloplasmin and copper/albumin ratios in urine were consistently greater than those in serum which were not different between patients and healthy controls except the copper/albumin ratio in macroalbuminuric patients. The ratios in urine decreased in parallel with the progression of nephropathy. Copper was found to be released from ceruloplasmin under acidic conditions. Conclusion: Urinary copper excretion in healthy controls may be the result of dissociation from the albumin-copper complex of serum during its passage through the kidney. In diabetic patients with advanced nephropathy, urinary copper excretion may be due to dissociations from both copper-albumin and ceruloplasmin-copper complexes filtered through the damaged glomerulus. Overloading of urinary copper to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy.

Apoptotic degeneration in the arhinencephalic brain of the mouse mutant Pdn/Pdn.

The homozygotes of a mouse strain with genetic polydactyly (Polydactyly Nagoya, Pdn) exhibit arhinencephaly and various brain malformations. In the present experiment, abnormal apoptotic degeneration in the arhinencephalic brain of Pdn/Pdn embryos and newborns was investigated immunohistochemically and by molecular genetic techniques. Polyclonal antibody against single-stranded DNA detected the nuclei of programmed dying cells (apoptotic cells) specifically in the interdigital necrotic zone of the normal mouse limb plate on day 14 of gestation. We used this antibody against single-stranded DNA to investigate the apoptotic degeneration in Pdn/Pdn brain. Abnormal apoptosis was observed in the infralimbic cortical plate, hypothalamus and periventricular thalamus on day 0 after birth in Pdn/Pdn brains. The TRPM-2 gene, which has been considered to mediate apoptosis, was expressed in the developing normal and Pdn/Pdn brains. TRPM-2 gene expressions in the brain stem and cerebellum of arhinencephalic Pdn/Pdn fetuses and newborns were higher than those of +/+ littermates. From these facts, it was suggested that the abnormal apoptosis caused a large amount of cell loss in the arhinencephalic mouse brain, and this cell loss induced the expansion of the ventricle, followed by the hydrocephaly.

Enhancement of TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method using mung bean nuclease, a single-stranded DNA digestion enzyme.:

The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method has been employed widely to demonstrate apoptotic cells in routinely prepared paraffin sections. Because the apoptotic cells were reactive with the antibody to single-stranded DNA, we attempted to enhance the TUNEL positivity by pretreatment with single-stranded DNA digestion enzymes, S1 nuclease, and mung bean nuclease. When mung bean nuclease (5 U/50 microliter/section) was incubated at 37 degrees C for 30 min, the TUNEL reaction was most effectively enhanced. Pretreatment with S1 nuclease (0.25 U/50 microliter/section) at 37 degrees C for 45 min was less reliable. Compared with the conventional TUNEL sequence, the enhancement technique using mung bean nuclease enabled us to detect more apoptotic cells in human small intestine, colon, tonsil, thymus, endometrium, ovary, liver, kidney, and pancreas. The positivity was not affected by autolytic change. The mechanism of enhancement is discussed.

The effect of silver administration on the biosynthesis and the molecular properties of rat ceruloplasmin

To examine the cause of the altered ceruloplasmin (Cp) metabolism by silver administration, we analysed the properties of serum Cp by gel filtration chromatography, affinity chromatography and polyacrylamide gel electrophoresis. Metal contents in the Cp fraction from the silver-treated group were estimated as approximately 0.8 atom of silver and 4.2 atoms of copper per molecule, and as 5.9 atoms of copper for the control group. These findings confirm that holo-Cp from rat serum administered with silver nitrate exists as a silver-bound inactive form, suggesting that silver displaces one of Cps copper atoms associating with oxidase activity. Matured holo-Cp also appeared in the Golgi in both groups, however, the amounts of enzymatically active holo-Cp showed a decrease after silver administration, while the apo-Cp level was hardly changed. These findings suggest that silver-bound holo-Cp is accomplished at Golgi.

Detection of DNA Fragmentation in Human Breast Cancer Tissue by an Antibody Specific to Single-stranded DNA.

While there have been many reports concerning the clinical significance of bcl-2 expression in human breast cancer, little is known about apoptosis in primary breast cancers. We immunohistochemically examined DNA fragmentation in 107 primary human breast cancers from Japanese women using an antibody specific to single-stranded DNA. The apoptosis index, calculated as the product of the positive cell number and the cellularity coefficient, ranged from 0 to 48. The average incidence of apoptosis was calculated as 0.1% of tumor cells. No relationships were observed among the apoptosis index, expression of bcl-2, and the histological grade of the tumors. Almost all apoptotic cells were phagocytosed by surrounding tumor cells immediately after DNA fragmentation. Apoptotic body formation was rare. The apoptotic cells seemed to be degraded within phagocytes, leaving no trace of apoptosis except the tiny shells of nuclei. The intensive phagocytic reaction might be one of the main reasons for the low incidence of apoptosis in human breast cancers.

Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats

Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.

Behavioral effect of mouse fibrinopeptide A on mouse forced swimming

A specific dopamine 2 receptor antagonist, (-)sulpiride, induced an anti-depressive behavior, climbing, in mice forced to swim for 6 h after the injection. The effective fraction was divided from the mouse serum using an ion exchanger and an ultra filtration method. This fraction contained fibrinopeptide A. A peptide synthesized according to the primary 6-amino acid sequence (TDTEDK) of fibrinopeptide A also remarkably increased the behavior. The present findings clearly indicate that a peptide with TDTEDK showed anti-depressive activity.