Melting temperature analysis as quantitative method for detection of point mutations.

Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K-ras gene in DNA of subjects with colorectal cancer. The K-ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K-ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.

Expression of p185 and p53 in benign and malignant colorectal lesions.

The c-erbB2 gene has been found to be amplified in a number of human adenocarcinomas, leading to elevated levels of expression of its encoded product, p185. Mutations in the p53 gene are also common in colorectal carcinomas, brain tumours, leukaemia and lymphomas. In this study, p185 and p53 overexpression was analyzed in colorectal adenomas (22 tubular adenomas and 2 tubulo-villous adenomas) and moderately differentiated adenocarcinomas (n = 22) in order to determine whether there was a relationship between these two proteins. The proteins are encoded by two genes located in the same chromosome. p185 and p53 expression was determined on tissue sections by immunohistochemical staining procedure. Expression of p185 was significantly higher (p < 0.01) in preneoplastic lesions (95.8% of cases) than colorectal cancer (63.6% of cases). p53 showed an inverse pattern to p185, being expressed in 58.3% of benign lesions and 72.7% of adenocarcinomas. These results confirm that p185 overexpression is associated with the early stages of colorectal cancer, whereas p53 is associated with more advanced stages. Although there was no correlation between p185 and p53 expression in premalignant lesions and adenocarcinomas, these two proteins have an important role in the adenoma-carcinoma sequence.

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after treatment with oxonic acid and allopurinol.

In our previous experiments on rat liver we found that 15' after intraperitoneal administration of 14C-formate the specific radioactivity of allantoin was always higher than that of uric acid. The present experiments have been carried out to interpret this unexpected result, which was only observed in liver and we studied: a) the incorporation of 14C-glycine into uric acid and allantoin; b) the effects of two competitive inhibitors of xanthine oxidase and uricase, oxonic acid and allopurinol respectively, on levels of uric acid and allantoin in liver and on their specific radioactivity after administration of labelled precursor. The results suggested: a) that under normal conditions, the formation of allantoin is so fast that it exceedes export from liver to serum, and thus the radioactivity of labelled precursors accumulates in allantoin; b) that when allopurinol or oxonic acid are administered, the rate of export exceeds that of allantoin formation and the incorporation of radioactivity into allantoin is lower; c) that not all the data, however, could be interpreted on this basis, but seems to require the existence of different pools of uric acid, which are transformed separately into allantoin.

Effect of estradiol on serum triglyceride lipoprotein levels and fatty acid composition in castrated rats.

Triglyceride content and fatty acid composition of rat serum lipoproteins showed specific variations after castration and estradiol treatment. Triglyceride levels decreased in VLDL after castration and in LDL and HDL after low doses of estradiol. High doses of estradiol enhanced triglyceride levels in VLDL and decreased them in LDL. Fatty acid composition showed a complex pattern: after castration, monoenoic acids decreased and essential fatty acids increased in all lipoprotein classes, enhancing the EFA/NEFA and EFA/ME ratios. Both doses of estrogen lowered these ratios in VLDL and LDL, but decreased them in HDL with high doses and enhanced them in HDL with low doses.

Effect of estradiol on phospholipid lipoprotein levels and fatty acid composition in the rat.

Phospholipid content and fatty acid composition in the different serum lipoproteins showed specific variations after castration and estradiol administration. Only the levels of phospholipids in HDL, the principal lipoprotein carrying phospholipids, increased after castration and were further enhanced by estradiol treatment, especially at low doses. Fatty acid composition showed many variations and an irregular pattern. The EFA/NEFA, EFA/ME and SAT/ME ratios were calculated. EFA/ME increased in VLDL after both doses of estradiol, while EFA/NEFA and EFA/ME of LDL enhanced at the low dose and decreased at the high one in a dose-dependent manner. HDL showed higher EFA/ME and SAT/ME ratios after castration and lower values of all ratios after both doses of estradiol. The correlation with diseases more frequent in men is discussed.

Labelling of uric acid and allantoin in different purine organs and urine of the rat.

We studied the incorporation of 14C-formate into uric acid and allantoin in different organs (liver, lung, kidney, spleen), isolated hepatocytes, perfused liver and urine of the rat. Allantoin had a higher specific radioactivity than uric acid after 14C-formate load in the liver in vivo. This was found to be a strictly hepatic phenomenon and not due to the influence of other tissues.

Levels of folic acid in plasma and in red blood cells of colorectal cancer patients.

The levels of folic acid have been determined by radioimmunological method in the plasma and in the red blood cells of normal subjects and colorectal cancer patients. A decrease was evident both in the plasma and erythrocytes of cancer patients. The possible reasons and applications of this observation are discussed.

Separation and determination of liver uric acid and allantoin.

We previously described the only satisfactory procedure yet achieved for separating uric acid and allantoin from rat liver. The procedure was based on trichloroacetic acid (TCA) extraction, acid hydrolysis, treatment with Hg-acetate, and cation- and anion-exchange chromatography. After separation, allantoin was quantified by a colorimetric method, and uric acid enzymatically using uricase. Since this procedure is too time-consuming, we propose an improved version which avoids the need for anion-exchange chromatography and the complex assay of catabolic compounds. The new method consists of a very fast and simple HPLC separation and direct determination of uric acid and allantoin at 220 nm. The method can be used for fresh tissue or after treatment of the tissue with labeled precursor.

Influence of estrogen on cholesterol esterification and fatty acid composition in serum lipoproteins of castrated rats.

Total, free and esterified cholesterol and its fatty acid composition were measured in the serum lipoproteins of castrated rats after estradiol administration. In general, castration and treatment with estradiol led to a decrease in total esterified cholesterol content. However, estradiol induced an effect opposite to that of castration on the fatty acid composition in VLDL. The effects were variable in HDL and insignificant in LDL. Similarly, the ratios of essential fatty acid to non-essential fatty acid (EFA/NEFA) and that to monoenoic acid (EFA/ME) were affected differently in castration and estradiol treatment in VLDL, but not in HDL or LDL. The pattern of lipid metabolism in castrated and estradiol-treated rats thus appears opposite to that described in human pathology.