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Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
2To whom correspondence should be addressed. E-mail: thliao{at}ccms.ntu.edu.tw.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • DNase I • parotid gland • small intestine • starvation • refeeding
DNase I (EC 3.1.21.1.) requires Mg2+ or Mn2+ for its catalytic activity, has an optimal pH of 
7–8 and produces 5'-phosphate nucleotides on hydrolysis of DNA. DNase I has been found mainly in the pancreas of most species of vertebrates studied (1
). Bovine pancreatic DNase I is the most thoroughly studied DNase I and consists of a single polypeptide with a molecular weight of 31,000 (2
). However, in rats DNase I is found mainly in the secretary cells of the parotid gland and not in the pancreas. The small intestine of rats also expresses a substantial amount of DNase I in the enterocytes covering the villi (3
,4
). The cDNA sequences of human, rat and mouse DNases I have been cloned and sequenced (5
–7
). These available cDNA sequences facilitated our studies on the regulation of DNase I at the gene expression level under various physiological conditions.
DNase I has been implicated in programmed cell death (apoptosis) (8
) and has been studied intensively in this field. However, DNase I as a digestive enzyme that is physiologically regulated is poorly understood. Although nucleotides are not essential nutrients as are some amino acids, several studies have suggested that dietary nucleotides appear to be important in supporting cellular metabolism and function, particularly in rapidly dividing tissues such as lymph and intestine (9
–11
). Nucleotides are present in regular diets, and an adult consumes 1–2 g of nucleotides/d in normal food (12
) in the form of DNA or RNA. Nucleases are required to degrade DNA and RNA before they can be absorbed by the body. DNase I is mainly responsible for the digestion of dietary DNA. In this study we investigated the regulation of the DNase I activities and the levels of their mRNA expression in the parotid gland and the small intestine during starvation and refeeding in rats.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar rats weighing 160–170 g, obtained from the Animal Center of the National Taiwan University (Taipei, Taiwan), were housed in groups of three or four in stainless steel cages on a 12-h light cycle (0700–1900 h) with free access to a nonpurified diet (Purina Certified Rodent Chow 5001; Ralston-Purina, St. Louis, MO) and tap water for 10 d before the experiments. Rats were not fed for 48 h, were fed nonpurified diet at 0900 h for 2 h and were killed in a CO2 chamber at 0, 0.33, 0.67, 1, 2, 6 or 12 h after refeeding. Experimental groups consisted of three rats except there were four rats in the control group. In a separate experiment, nonpurified diet in a sealed transparent flask was placed in the cage of the starved rats, with the diet visible but not available for eating. These rats were killed 30 and 60 min later. Water was given ad libitum during all the experiments. Immediately after the rats were killed, parotid glands and a 2-cm segment of the small intestine from each rat were removed from the part just distal to the stomach. The tissues were frozen immediately in liquid nitrogen and stored at -70°C until measurements for DNase I activity and the amount of mRNA. All animal experimental procedures followed the "Guide for the Care and Use of Laboratory Animals" of the National Science Council, Taiwan."
DNase I assay.
Parotid gland or small intestine tissue was homogenized in cold buffer containing 0.1 mol of Tris-HCl (pH 7.0) and 20 mmol of CaCl2 per L and centrifuged for 20 min at 13,000 x g; the supernatant was kept for activity assay. DNase I activity in the parotid gland was determined by the hyperchromicity assay as described (13
). DNase I activity in the small intestine was relatively low and was determined by a fluorescence assay. This high sensitivity assay method was carried out as follows. Crude extract of the small intestine containing 300 µg of protein was added to each well of a 96-well Corning plate that contained 0.25 µmol of ethidium bromide, and 2 g of DNA in reaction buffer [100 mmol of Tris-HCl (pH 7.2), 10 mmol of CaCl2, 10 mmol of MnCl2 and 500 mg of bovine serum albumin per L] per L in a total volume of 200 µL. Fluorescence was measured every 150 s with excitation at 485 nm and emission at 645 nm on a CytoFluor 2300 Fluorimeter (Millipore, Bedford, MA). The fluorescence intensities were plotted against time. These kinetic data plotted with the data from the hyperchromicity assay were used for presentation of DNase I activity. For calculation of specific activities, the protein amounts were determined by the Bradford method (14
) with bovine serum albumin as the standard.
Total RNA extraction and slot-blot analysis.
Equal amounts of parotid or small intestine tissue from three rats at each time point were pooled for extraction of RNA. Total RNA was isolated according to the method of Chomczynski and Sacchi (15
). Northern blot or slot-blot analysis was performed following standard methods (16
) using a biotin-dCTP labeled rat DNase I cDNA as probe and was detected using a Southern-Light Chemiluminescent Detection System (Tropix, Bedford, MA). A 763-bp rat DNase I cDNA fragment and a 204-bp rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH)3 cDNA fragment were amplified by reverse transcriptionpolymerase chain reaction from rat parotid RNA using specific primers designed according to the published sequences (7
,17
). The amplified DNA fragments were cloned into a pGEM-Teasy vector (Promega, Madison, WI) and sequenced to confirm their identity. The signals of DNase I and GAPDH were quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The amount of DNase I mRNA was normalized to that of GAPDHmRNA and expressed relative to those in the starved control rats (relative value = 1).
Statistical analysis.
Results were expressed as means ± SD. Comparisons were made using Student’s t test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DNase I activity in the parotid gland of rats that had been deprived of food for 48 h was 2.2-fold that of the fed control rats (P < 0.05) (Table 1
). The activity decreased very quickly in response to feeding. A 34% decrease in DNase I activity (P < 0.05) was detected 20 min after refeeding, whereas the lowest activity (33% of starved controls, P < 0.05) occurred 1 h after refeeding. DNase I activity increased gradually after that and reached the level of fed control rats at 6 and 12 h. Samples analyzed by the zymogram method revealed a DNase I activity band of 31 kDa corresponding to the purified bovine DNase I, confirming that the activity measured using the hyperchromicity assay was DNase I and was not due to other DNases (data not shown).
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. The activity increased gradually after feeding. A 39% increase in DNase I activity (P < 0.05) occurred 40 min after refeeding and reached a peak (211% of starved control levels, P < 0.05) at 2 h. The activity in the small intestine decreased gradually after food was removed and reached the basal level 10 h later. Expression of DNase I mRNA in the parotid and the small intestine.
Northern blot analysis detected a single band of DNase I mRNA, with the highest amount in the parotid gland and a smaller amount in the small intestine, and a barely detectable signal in the submaxillary gland and kidney (data not shown). The mRNA was not detected in the liver, stomach, pancreas, lung, heart, skeletal muscle, spleen, adipose, large intestine, lymph nodes, thymus or brain by Northern blot analysis. However, it was detected in the spleen, liver, lymph nodes and thymus by reverse transcription polymerase chain reaction (data not shown).
The effects of starvation and refeeding on the levels of DNase I mRNA were determined in pools from three rats by slot-blot analysis. Although starvation led to the accumulation of DNase I activity in the parotid gland, only a small amount of DNase I mRNA could be detected during this time (Fig. 1
). In response to feeding, DNase I mRNA increased very quickly and reached a peak of 12-fold that of starved control rats at 40–60 min. The mRNA decreased 25% from the peak in the next 1 h and then remained near a constant amount of 80% that of the peak value during the next 10 h. Thus in the parotid gland, the changes in DNase I mRNA occurred in reverse of those in the DNase I activity.
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). Although the response was not as fast as that in the parotid gland, intestinal DNase I mRNA reached a peak value of 9.9-fold that of starved controls at 1 h after refeeding. The amount of mRNA declined very quickly and returned to the basal level 10 h later.
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Food-deprived rats offered diets in a transparent flask had a 19% decrease (P = 0.05) of DNase I activity in the parotid gland in 30 min (from 37.7 ± 1.0 to 30.5 ± 2.2 U/mg protein). However DNase I mRNA increased 2.5-fold, and the amount of mRNA decreased to a concentration even lower than that of the control rats at 1 h (Fig. 3
).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) have suggested that expression of DNase I in the small intestine and kidney is involved in cellular turnover and programmed cell death for these tissues. It is not clear whether DNase I is secreted into the alimentary tract from the small intestine. However, our data showed that intestinal DNase I activity and mRNA were clearly up-regulated by feeding. These data suggested that DNase I in the small intestine not only was involved in the apoptotic pathway but also might play an important role as a digestive enzyme. DNase I in the parotid gland at a certain time point represents the balance of the secretion and synthesis of the enzyme. Therefore, a decrease in the enzyme activity in the tissue does not mean that synthesis of the enzyme is also decreased. There probably is a storage pool of DNase I in the parotid gland. The enzyme in this pool is secreted with saliva, resulting in a decrease in enzyme in the gland while eating. However, at the same time the amount of mRNA increases in the gland, suggesting that the synthesis of the enzyme is induced by feeding. It is possible that the secretion rate was higher than the synthesis rate for the enzyme, resulting in the decrease of DNase I in the parotid gland. In the small intestine, there probably is no storage pool of DNase I; the enzyme and mRNA of DNase I were low after 48 h of food deprivation, and the synthesis of the mRNA and protein was stimulated in response to feeding.
Sreebny and Johnson (18
) have demonstrated that mastication plays an important role in regulating the synthesis of the secretary products of the parotid gland. In this study, we found that DNase I mRNA was up-regulated in rat parotid by feeding, and mastication might not be essential for the regulation because the amount of mRNA was increased 2.5-fold within 30 min when the diet was not offered but was seen in a sealed transparent flask (Fig. 3)
. It has been demonstrated that the synthesis and secretion of amylase in rat parotid gland are accelerated by parasympathetic and sympathetic nerve stimulation in vivo (19
). It is possible that the synthesis and secretion of DNase I in parotid gland were also regulated, at least in part, by the nerve impulses.
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FOOTNOTES |
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3 Abbreviation used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Manuscript received 31 August 2002. Initial review completed 29 September 2002. Revision accepted 9 October 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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