Jukka Tenhunen

Characteristics of catechol O-methyltransferase (COMT) and properties of selective COMT inhibitors

The enzyme-catalyzed O-methylation of catecholamines was first described by Axelrod and coworkers in the late 1950’s [1–3]. They called the responsible enzyme catechol O-methyltransferase (COMT). During the subsequent 15 years the enzyme was partially purified, its distribution was established, several reaction mechanisms were proposed, and a number of inhibitors were described. The results of this study period were extensively reviewed by Guldberg and Marsden in 1975 [4].

Hydrolysis of single-stranded RNA in aqueous solutions―effect on quantitative hybridizations

Single stranded RNA is non-enzymatically hydrolysed in aqueous solutions at neutral pH at elevated temperatures. This hydrolysis causes practical problems in different hybridization procedures. Synthetic cRNA transcribed from the human p53 oncogene was found to be partially destroyed after 6 hours and completely destroyed after overnight incubation at 60 degrees C. At lower temperatures the cRNA was preserved intact in spite of overnight incubation, but at higher temperatures (80 degrees C) the degradation occurred in less than 2 hours. The effect of the hydrolysis on hybridization results was studied by measuring in solution the hybridization kinetics of the cRNAs of another human oncogene, N-myc. It is obvious that conditions generally used for DNA hybridizations cannot be utilized for RNA in quantitative studies.

A solution hybridization method for quantification of mRNAs: Determining the amount and stability of oncogene mRNA

A solution hybridization method for the quantification of specific mRNAs is described. This assay utilizes complementary RNA probes prepared by in vitro transcription, sandwich hybridization in solution, and affinity-based hybrid collection. The possibility of using this method for crude biological samples without purifying mRNAs makes it ideal when accurate quantification of multiple samples is needed. Human N-myc oncogene transcript was used as a model and as little as 0.24 pg (2 X 10(5) molecules) of N-myc mRNA could be detected. Using this assay it was shown that human neuroblastoma IMR-32 cells contain approximately 500 N-myc mRNA molecules per cell having a half-life of approximately 35 min.

Screening of premalignant cervical lesions for HPV 16 DNA by sandwich and in situ hybridization techniques

A series of 97 cervical smears and 69 directed punch biopsies derived from 84 consecutive women prospectively followed-up for cervical HPV (human papillomavirus) infections were studied using the sandwich hybridization and in situ hybridization techniques with HPV 16 DNA probes. The aim was to test the sensitivity and applicability of these two techniques in routine diagnosis of cervical HPV infections from smears. As a measure of specimen adequacy, the number of cells recovered in the cervical scrape was determined along with HPV 16 DNA in the sandwich hybridization test using human pro-alpha 2(I)-collagen gene probe. CIN (cervical intraepithelial neoplasia) was suggested in 56% of the patients by the Pap smear, and disclosed in 65% of the biopsies. HPV 16 DNA was present in 57% of cervical scrapes consistent with CIN, i.e., were of Pap smear classes III or IV. Forty percent of the scrapes not suggestive of CIN, i.e., Pap smear classes I or II, also contained HPV 16 DNA. The detection rate for HPV 16 DNA of the sandwich hybridization method was 89% of that of the in situ method in adequate scrapes, but only 43% in cell-poor specimens. The number of HPV 16 DNA-positive scrapes as compared with the total number of diagnoses obtained by studying also the biopsies was 31/36 (69 patients). The results indicate that the cervical scrape as a noninvasive specimen is applicable for screening of cervical HPV infections, and it can be studied with acceptable sensitivity by the rapid sandwich hybridization technique. However, if a punch biopsy is indicated it should be studied using the in situ hybridization technique that allows more sensitive detection of HPV DNA than any other hybridization method and enables the analysis of several HPV types in the same sample instead of only one HPV type in the scrapes.