Bernard Shapiro

Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease.

Previous studies showed that patients with neoplasms of various types and origins have abnormally high concentration of DNA in their serum. The current work compares circulating DNA levels in patients with benign or malignant disease of the gastrointestinal tract and determines the diagnostic value of such measurements. DNA was quantitated by radioimmunoassay capable of detecting 25 ng/ml, and as a simple and noninvasive test, it could be a useful addition to other diagnostic procedures. The GI tract was chosen because it affords a comparison of benign, precancerous, and malignant lesions of the same organ. Of the 386 patients studied prospectively, 48% had benign disease and mean DNA levels (±SE) of 118 ± 14 ng/ml, whereas 52% had malignant disease and 412 ± 63 ng DNA/ml. The difference was statistically significant (P < 0.001). The DNA assay showed the highest sensitivity for pancreas carcinoma: 90% of the patients had DNA levels above 100 ng/ml, chosen as the upper normal limit. Simultaneous measurements of both DNA and carcinoembryonic antigen (CEA) resulted in increased sensitivity and specificity, even when either marker alone had low sensitivity (gastric carcinoma). The results indicate that serum DNA concentration is markedly elevated in malignancy, and moderately elevated in benign disease, as compared with normal controls. These findings may have diagnostic and prognostic value.

The nature of the membrane injury in irradiated human erythrocytes.

The mechanism of the radiation-induced increase in sodium accumulation of erythrocytes was studied by chemically altering the cell surface and the cell interior. Agents such as PCMBS and GED, which block surface sulfhydryl groups, mimicked the radiation effect but, when employed with radiation, reduced the radiation effect. Papain, which increased the surface sulfhydryl groups, increased the radiation effect. Trypsin and neuraminidase, which did not alter surface sulfhydryl groups, did not alter the radiation effect. Agents that altered the heme (nitrite, carbon monoxide, nitrogen) or globin (

Radioimmunoassay for nanogram quantities of DNA

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The Mechanism of Radiation Hemolysis in Human Erythrocytes

) of hemoglobin inside the cells did not alter the radiation effect. The measurement of sulfhydryl groups on the cells showed decrease on irradiation. Reversal of PCMBS effect on sodium accumulation was observed with GSH and MEG and correlated with the removal by these agents of a small amount of the PCMBS from the cells. Reversal of of radiation effect on sodium accumulation was observed with B...

A comparison of DNA and DNA-binding protein levels in malignant disease

A direct competitive binding radioimmunoassay for DNA has been developed, using 125I-iododeoxyuridine-labelled DNA as the antigen and the serum from a patient with systemic lupus erythematosus. The assay is sensitive in the range of 25 to 1000 ng/ml of DNA. The sensitivity is determined by the affinity of the antibody: this SLE serum contains a component with an association constant of 9.6 X 10(-5) l/mol active at high dilution (1/10,000). Any biological material, such as serum, synovial fluid or tissue extracts can be tested directly. No interference has been found by DNAse in normal serum, or inhibition by mononucleotides or RNA. Native or denatured DNA from different sources (Escherichia coli, salmon sperm, calf thymus and human placenta) either purified or not, competes equally well for the antibody in this system.

The Mechanism of Action of AET I. The Radiation Chemistry of 2-Mercaptoethylguanidine and Bis- (2-guanidoethyl) Disulfide in Aqueous Buffered Solutions'

Human bank blood erythrocytes were given hemolyzing (16 to 50 kR) exposures of60 Co gamma rays, suspended in various media, and maintained at 5°C for 20 hours. Hemolysis occurred in isotonic NaCl but was prevented or reduced in isotonic choline chloride and in hypertonic NaCl or KCl. Prior to hemolysis in isotonic NaCl, the cells rapidly lost potassium and accumulated sodium. Although irradiated suspensions of cells in choline chloride did not hemolyze, they lost potassium. 2-Mercaptoethylguanidine (MEG) and reduced glutathione (GSH), which does not enter cells, reduced hemolysis and potassium loss when added to the suspensions before irradiation. Radiation caused a marked decrease in membrane sulfhydryl groups. Blockage of membrane sulfhydryl groups by p-chloromercuribenzoate (FCMB) and p-chloromercuribenzene sulfonate (PCMBS), which does not enter the cells, mimicked radiation by causing potassium loss and hemolysis. PCMBS-induced hemolysis was prevented by suspension of the cells in isotonic choline ch...

The mechanism of action of AET. IV. The distribution and the chemical forms of 2-mercaptoethylguanidine and bis(2-guanidoethyl) disulfide in protected mice.

Abstract Measurements of serum concentrations of DNA and the DNA-binding protein C3DP by radioimmunoassay showed that the levels of both substances tend to increase in cancer patients during active malignant disease. In most cases, the levels returned to normal during chemotherapy-induced remission; however, the changes in concentration for DNA and C3DP did not occur simultaneously, and no correlation was found between their levels. The sera of cancer patients contained a strong inhibitor of DNAse. We examined the possibility that C3DP may have such an inhibitory effect by binding to DNA and preventing the action of DNAse. The enzyme was fully active in the presence of purified C3DP, indicating that the DNAse inhibitor in cancer serum was a substance other than C3DP. Although the relationship between DNA and the DNA-binding protein remains unknown, their measurement may have diagnostic and prognostic value.

The protection of mice against radiation by 2-mercaptoethylguanidine and its disulfide.

Issued by School of Aviation Medicine, Brooks AFB, Tex. Aqueous buffered solutions of MEG and GED were irradiated with a 3-curie Co/sup 6//sup 0/ gamma source. The radiation products were separated and identified by paper chromatography and electrophoresis. Irradiation of solutions of MEG produced GED. Irradiation of solutions of GED produced 2-gusnidoethanesulfinic acid, taurocyamine, inorganic sulfate, and guanidine. G values were calculated for the oxidation of MEG at pH 6 (14.3 to 18.2), and for the oxidation of GED at pH 7.5 (0.29) and pH 8.5 (1.0). G values were also calculated for the formation of 2- gusnidoethanesulfinic acid plus taurocyamine at pH 7.5 (0.42), and for the formation of inorganic sulfate and guanidine at pH 7.5 (0.17). The oxidation of MEG and GED by ionizing radiation indicated that these protective agents were capable of reacting with oxidizing free radicals formed in irradiated aqueous solutions. (auth)

MECHANISM OF THE PROTECTIVE ACTION OF GED AGAINST RADIATION DAMAGE TO DNA.

The distribution and chemical nature of the protective agent in the various tissues of 8- to 10-week-old C57BL/6J male mice given a protective dose of MEG-S/sup 35/ and GEDS/sup 35/ were studied. Results were obtained at 20 minutes after an intraperitoneal (i.p.) injection of 140 and 280 mg of MEG per kilogram and at 20, 60, and 120 min after an i.p, injection of 140 mg of GED per kilogram. Protein-bound S/sup 35/, GED, taurocyamine, guanidoethanesulfinic acid, MEG, and sulfate were identified in the serum, tissues, and excreta. Several unknown compounds were also observed. Concentrations of protein-bound S/ sup 35/ and GED decreased at 120 minutes after GED administration and correlated best with protection of the mice. All products increased in the tissues at the higher dose of MEG. Some differences in the concentrations of products were noted on comparison of MEG with GED-treated animals. The relation of the results to the mechanisms of action of MEG and GED is discussed. (auth)

The mechanism of action of AET. VI. The protection of proteins against ionizing radiation by GED.

Some sulfhydryl compounds lose their ability to protect against ionizing radiation when they are administered in the oxidized state. Oxidized glutathione (1, 2) and cystine (3), the oxidized form of cysteine, are inactive as protectors, whereas cystamine (4-7), the oxidized form of cysteamine, is highly effective in reducing the injurious effects of radiation. Gordy and Miyagawa (8) have suggested that cystamine is effective only on its reduction to cysteamine within the animal cell. 2-Mercaptoethylguanidine (MEG) is formed by transguanylation when S-(2aminoethyl)thiuronium bromide hydrobromide (AET) is dissolved in aqueous buffered solutions at neutral pH (9). Hence, MEG rather than AET has usually been the substance administered in previously reported protective studies. MEG is readily oxidized in mildly alkaline solutions to bis(2-guanidoethyl) disulfide (GED), and solutions of MEG are therefore frequently contaminated with the disulfide. In their survey of related compounds with protective activity, Shapira et al. (10) observed that GED, as well as MEG, protected mice against radiation. The present study was undertaken to investigate, in greater detail, the relationship between the state of oxidation of this agent prior to administration, and its therapeutic efficiency.