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The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washings

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Abstract

1. Sialic acid has been found to interfere with three colorimetric reactions used for the estimation of DNA: a modified diphenylamine reaction at 100 degrees (Dische, 1930), the nitrophenylhydrazine method (Webb & Levy, 1955) and the diphenylamine reaction at 30 degrees (Burton, 1956). 2. Evidence is presented that sialic acid is present in hydrolysates obtained from gastric wash-out material. 3. A mathematical method for correcting for interference from sialic acid in the diphenylamine reaction at 30 degrees is described. 4. The diphenylamine reaction has been modified to make it suitable for the estimation of DNA in the presence of sialic acid. The modifications are to increase the concentration of diphenylamine to 2% and to perform the reaction at 6-13 degrees for 48hr. These modifications increase the sensitivity 25% above Burton's (1956) modification of the diphenylamine reaction. 5. The precipitation, extraction and recovery of DNA from gastric wash-out material have been investigated.

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... The protein content was measured following the method of Lowry et al. [19] using bovine serum albumin as a standard. Nucleic acid concentrations were measured following the method of Croft-Lubran [20]. Sialic acid content was measured following the periodate-resorcinol method [21]. ...
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... The mucosal protein was determined by the method of Bensadoun and Weinsten (1976) and expressed as mg protein/g wet tissue. Mucosal DNA was extracted by serial precipitation with cold perchloric acid and assayed according to Burton (1956) as modified by Croft and Lubran (1965), using E. coli DNA as standard. DNA was expressed as µg/mg protein. ...
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The small bowel of hypophysectomized rats was examined histologically and chemically. The following conclusions were made:1. The diameter of the small bowel of hypophysectomized rats was smaller than that in normal controls. The villi were shorter, often broader and more blunt. Increased lymphocytic infiltration was noted in the jejunum, ileum, and colon. 2. The percent of weight of the entire small bowel in terms of total body weight was smaller in hypophysectomized rats. Both the dry and wet weights of the small bowel were significantly decreased in hypophysectomized rats. 3. No significant differences were found between the content of control and hypophysectomized animals with regard to protein, total nitrogen, phosphorus, P-RNA, and P-DNA of the small bowel, if the concentrations are expressed per gram of dry tissue. 4. Uronic acid and sialic acid were present in lesser amounts per gram of dry tissue as well as in the entire small bowel of hypophysectomized rats. By contrast, lipids were present in increased amounts. 5. All chemical components tested, except lipids, were present in lesser amounts in the entire small bowel of hypophysectomized rats.
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The purification of estrogen- and progesterone-binding proteins of human uterus by employing affinity resins coupled with steroid-bovine serum albumin conjugates, led to the isolation of preparations with estrogen- and progesterone-binding sites havingK d values in the range of 0.96 to 1.20 × 10-9 M. These were different from theK d values of 10-10 M and 10-8 M obtained for two types of binding sites present in the crude cytosolic and nuclear fractions. The purified proteins sedimented on sucrose gradient withS values in the range of 3.6–4.4. The cytosolic and nuclear estrogen- and progesterone-binding proteins, thus purified, showed differences in specificity of binding to the hormone. While the cytoplasmic proteins were more specific in their binding to estradiol or progesterone, the nuclear proteins bound Cortisol with equal or moderate affnity. These results demonstrate the presence of distinct physiological forms of estrogen- and progesterone-binding proteins in the cytoplasm and nucleus, thus pointing to the importance of both these compartments in hormone action.
Article
The distribution of14C-labelledm-AMSA was studied in rats and pigmented mice using whole body autoradiography. The agent rapidly disappeared from the blood, accumulating in significant amounts in large parenchymal organs, certain endocrine tissues, and the retina of the pigmented mouse eye. The hemopoietic and lymphoid tissues showed a moderate uptake of radioactivity with the highest concentration observed in the thymus. The autoradiograms indicated a rapid excretion of radioactivity via the liver, kidney and the glandular part of the gastric mucosa. The distribution pattern of label from14C-m-AMSA remained unaffected by pretreatment of animals with high dose (500 mg kg−1 b.w.) of cytosine arabinoside. Injection of unlabelledm-AMSA (7 mg kg−1 b.w.) to growing rats 24 h before sacrifice resulted in a highly significant (P<0.001) inhibition of3H-thymidine incorporation into the DNA of thymus and spleen. A less pronounced reduction was observed in the kidney, adrenal, lung and testes. The thymidine incorporation into the DNA of bone marrow was markedly suppressed when calculated per dry weight, but increased when related to the DNA content, suggesting early regeneration of the remaining cells. In contrast, no significant effects were observed on the DNA synthesis in small intestine and liver.
Article
Small-bowel resection leads to hyperplasia of the residual small intestine. However, the factors initiating small-bowel hyperplasia are not clearly understood, although oral intake either by direct contact with the small bowel or via hormonal or neurovascular factors has been suggested as the major stimulus. In order to determine whether oral intake is an obligatory prerequisite for small-intestinal hyperplasia, we compared rats one week after undergoing a 70-cm proximal intestinal resection with sham-operated animals. Resected, orally fed rats demonstrated small-intestinal hyperplasia, whereas resected and sham-operated intravenously alimented rats did not. There were no differences in gut weight, mucosal weight, mucosal protein, or DNA between resected or sham-operated intravenously alimented rats. These data provide direct experimental proof that oral intake is a necessary stimulus for small-intestinal hyperplasia after resection.
Article
This study provides information on the rates of DNA synthesis, sites of DNA synthesis, and DNA content of the avian salt gland during the osmoticstressing (plasma membrane synthesis) and destressing (plasma membrane turnover) cycle, in an effort to better understand the relationship of cell turnover to the initial events in plasma membrane amplification, differentiation, and turnover. The rate of DNA synthesis increases 12–24 h after the onset of osmotic stress, is maximal at about 24 h of osmotic stress, and decreases thereafter in fully stressed and destressed glands. The maximum DNA and protein content, and the maximum protein/DNA ratio are obtained after about 3 days of stress. Autoradiograms show that at 24 h of stress 70–80% of DNA synthesis occurs in connective tissue cells and 20–30% in parenchymal cells, but by 6 days of stress, synthesis occurs about equally in these cell groups. Because destressing is characterized by a large decrease in plasma membrane and in glandular protein, but by little DNA turnover or loss, the loss of plasma membrane is likely due to some type of cell dedifferentiation rather than cell turnover.
Article
The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10−4 M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56°C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10−4 M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10−4 M spermidine.
Article
Es wird ber eine Mikrobestimmung von DNA in biologischem Material durch gas-chromatographische Thymin-Analyse berichtet. Nach Aufschlu der lyophilisierten Gewebe mit heier Ameisensure, Trocknen, Extraktion von Thymin mit Aceton/Wasser 982 (v/v), Sublimation und Dnnschicht-Chromatographie wird der Thymingehalt gas-chromatographisch bestimmt. Die Ausbeuten werden durch Isotopenverdnnungsanalyse kontrolliert. Die kleinste, mit dieser Methode nachweisbare DNA-Menge in Geweben betrgt etwa 10 g. Die relative Standardabweichung liegt bei 13%. Die gas-chromatographischen Ergebnisse lieen sich durch UV-Spektroskopie besttigen.A micro- determination of DNA in biological materials by gas-chromatographic analysis of thymine is reported. After decomposition of lyophilized tissues by heating with formic acid, drying, extraction of thymine with acetone/water 982 (v/v), sublimation and thin-layer chromatography the thymine content is determined by gas-chromatography, yields being controlled by isotope dilution techniques. The smallest amount of tissue DNA quantitatively detectable by this method is 10 g. The relative standard deviation is 13%. The gas-chromatographic results were monitored by UV-spectroscopy.
Article
The role of extracellular matrix of retinal pigment epithelial cells (RPE-ECM) in the regulation of the survival of choriocapillaris endothelial cells (CCE) was investigated in vitro. The CCE survival was evaluated by trypan blue staining, neutral red uptake, and the counting of viable cells. Results showed that CCE cells survived on RPE-ECM. Pre-treatment of RPE-ECM individually with neutralizing antibodies to acidic fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, or transforming growth factor β(pan specific to TGFβ1, TGFβ1.2, TGFβ2 and TGFβ5), did not alter the survival rate of CCE cells on RPE-ECM, as compared to that of the control (CCE survival rates on RPE-ECM pretreated with normal rabbit IgG). However, the treatment of RPE-ECM with neutralizing antibody to basic fibroblast growth factor (bFGF) caused CCE death by 77.1±15.7%. The CCE death was defined as apoptosis based on the morphological markers (shrinkage in cell size with blebbing of plasma membranes, condensation and fragmentation of nuclei, and DNA fragmentation in multiples of approximately 200 bp). The addition of phorbol 12-myristate 13-acetate (PMA) (2 nM) to the culture medium was effective for complete prevention of CCE apoptosis; the protecting effect of PMA on CCE apoptosis can be abolished by H7 (25 μM), but not HA1004 (50 μM), suggesting the involvement of PKC in protecting CCE from apoptosis. The inhibition of protein synthesis of CCE cells by cycloheximide (0.1 μM) did not affect the apoptotic process of the cells.In a separate experiment, when CCE cells were cultured in a medium saturated with bFGF (5 ng ml-1) without RPE-ECM, the cells also died by apoptosis. However, this apoptotic process was not affected by PMA. Cycloheximide also failed to affect the apoptotic process.These results suggest that both RPE-ECM insoluble molecules and RPE-ECM-bound bFGF modulate choriocapillaris survival by suppressing CCE apoptosis.
Article
Beef glomerular basement membranes, solubilized by sodium dodecylsulfate and , were examined by disc gel electrophoresis and gel filtration. The beef glomeruli were sonicated in either 0.15 m NaCl or 1.0 M NaCl, and the basement membranes sedimented at 1300 × g. Disc gel electrophoresis in sodium dodecylsulfate showed that the 1.0 M preparation contaiend predominantly high molecular weight components; the 0.15 M preparation contained moer low molecular weight components. Different amounts of hydroxyproline and DNA were found in the two membrane preparations. Other subunits were obtained when basement membrane was solubilized in 8 M urea or 7 M guanidine thiocyanate.Gel filtration of solubilized glomerular basement membrane was performed on 6% Agarose in sodium dodecylsulfate-phosphate. Partial separation of teh components observed in the disc gel electrophoresis was achieved. Protein and carbohydrate were present in all fractions.
Article
The macroscopic and microscopical appearances of human gastric washouts obtained after the administration of dispersions of soluble and pure calcium aspirin are described. Cytological evidence that these dispersions cause exfoliation of gastric epithelial cells is given. A quantitative method for estimating this exfoliation is described and the results of 21 tests are given. The deoxyribonucleic acid (D.N.A.) content of gastric washouts after aspirin has been observed to be significantly higher than control D.N.A. values in 10 of the 12 tests in which satisfactory control levels were obtained. In 9 of these 12 tests the rate of accumulation of D.N.A. in the stomach after aspirin was significantly higher than the control rate. This increased D.N.A. content and rate of accumulation of D.N.A. in the stomach after the administration of aspirin is considered to represent exfoliation of gastric epithelial cells. © 1963, British Medical Journal Publishing Group. All rights reserved.
Article
THE use of DNA (deoxyribonucleic acid) as a reference standard in biochemical investigations1, which is now well established, rests on the observed constancy of DNA content in each somatic cell in a species. With certain exceptions, the number of cells in a sample may be related to the amount of DNA2.
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