Shalom A. Leon

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.

Radioimmunoassay for nanogram quantities of DNA

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.

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

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.

A comparison of methods for labelling DNA for use in the radioimmuno assay of DNA-antibodies

Abstract The radioimmunoassay for DNA-antibodies in systemic lupus erythematosus is assessed with DNA labelled with 125 I via chemical iodination on one hand ( 125 I-DNA), and with DNA containing [ 125 I]iododeoxyuridine via biological incorporation on the other ([ 125 IUdR]DNA). The results show that chemical iodination labels protein impurities in the DNA and reduces the difference in binding of normal vs lupus sera. 125 IUdR-DNA is a superior product since no label is introduced in proteins. The binding of 125 I-DNA by normal sera ranges from 5 to 20% and from 40 to 70% lupus sera. The same sera yield values of 0–3% for normal and 92–98% for lupus with 125 IUdR-DNA. The latter allows a sharper discrimination between normal and slightly elevated values, as in patients under treatment.

Isolation of toxic subunits from two murine-toxic proteins from Pasteurella Pestis

Abstract Two mouse-toxic proteins, Toxin A (240,000 molecular weight) and Toxin B (120,000 molecular weight), were dissociated in sodium dodecyl sulfate to small molecular weight subunits. Subunit size from 10,000 to 12,000 molecular weight is proposed based on gel electrophoresis, ultracentrifugation and dialysis experiments. The subunits retained approximately 60% of the toxic activity of the large molecular weight toxins.

In Vitro Protection against Radiation Damage to Template Activity in DNA Synthesis

SummaryIn vitro protection against radiation-induced functional changes in DNA was studied by measuring DNA template activity in enzymatic DNA synthesis. Exposures up to 28 kR inhibited the priming activity of salmon sperm DNA with E. coli DNA-polymerase, reducing the incorporation of all four deoxynucleotides and altering the incorporation kinetics. The radiation protective agent bis(2-guanidoethyl) disulphide (GED) protected against these changes. GED-protected-DNA exposed to 7 and 14 kR actually showed enhanced template activity, incorporating 20–50 per cent more dGMP than the unirradiated control. Partial denaturation of DNA by dissolving in distilled water and complete denaturation by heat, before irradiation, increased the radiosensitivity of DNA. Heat-denaturation of unirradiated DNA did not affect its template activity, but heat-denaturation of DNA previously irradiated in double-stranded form reduced its template activity below that of irradiated undenatured DNA. Denaturation may release inactive...

Properties of DNA irradiated in the presence of the protective agent bis(2- guanidoethyl) disulphide (GED)

SummaryStructural alterations were measured after gamma-irradiation of dilute solutions of salmon-sperm DNA and were correlated with the enhancement of template activity at 7 kR, as well as its inhibition at higher exposures as previously described. Formation of single-stranded regions in irradiated DNA was demonstrated by measurements of hyperchromicity and Tm in the presence of formaldehyde, and by the rate and quantity of 14C-formaldehyde binding. The radioprotective agent GED showed little and marked protection against this damage at 7 kR and 28 kR, respectively. Ultracentrifugation analysis demonstrated the formation of double- and single-stranded breaks in a ratio of 1 : 6·5 without GED and 1 : 10 with GED at both 7 kR and 28 kR. It is concluded that enhancement of template activity at 7 kR is due to the production of single-stranded regions and single-stranded breaks which stimulate the activity of DNA polymerase. The protection by GED results in reducing the number of double-stranded breaks and ot...

Biosynthetic pattern of murine toxins in Pasteurella pestis cells and extracts

1. 1. The level of the protein toxins A and B within Pasteurella pestis cells is a function of the growth stage of the culture. Cells from midlog phase (27°) have minimal amounts of toxic material. Two generations later (early stationary phase) the toxic content has increased 7-to 10-fold. At this stage, the cells contain toxin amounting to 5–10 % of their total soluble protein. A small increase in toxin content is observed when the cells are grown at 37°. 2. 2. The low toxin content of young cell extracts is apparently not due to lower protein synthesizing capacity, since they are the most active in amino acid incorporation in vitro. Extracts from 37° grown cells on the other hand exhibit low amino acid incorporating activity, presumably due to limiting amounts of factor(s) in the soluble protein fraction. 3. 3. Pulse and pulse-chase experiments indicate that the two proteins are synthesized simultaneously and independently of one another. No interconversion between toxin A (mol. wt. 240 000) and toxin B (mol. wt. 120 000) by dissociation or dimerization, respectively, has been observed in the cells. 4. 4. Since toxic activity has been found associated with the membrane, and cells containing low levels of these proteins are quite fragile, the possibility that they constitute components of the cell envelope is discussed.