Densitometric determination of human papillomavirus DNA quantities by chromato-scanning in the fluorescence mode.

The quantitation of human papillomavirus DNA isolated from warts by chromato-scanning (fluorescence mode) photographs of ethidium bromide-stained agarose gels is described. Excitation at 200 nm (with a cutoff filter at 400 nm) generates fluorescence from the white portion of the printing paper. The fluorescent intensity correlated with the quantities of DNA in the band of interest. The amounts of DNA were determined using calibration curves of approximately the same size as lambda phage DNA fragments. This general method of quantification is applicable to photographs of other types of polynucleotides capable of being separated and stained in a gel medium.

Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods.

The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.

Expression of ras proto-oncogene related protein p21 in normal human skin and cutaneous tumours.

The cellular proto-oncogene, ras, is known to play an important role in the regulation of cell growth and proliferation in normal and malignant conditions. The present study was undertaken to immunohistochemically examine the expression of ras protooncogene product p21 in normal human skin and some cutaneous tumours. In normal skin, the expression of p21 was found in sweat glands, sebaceous glands, capillary endothelium, and smooth muscles, while epidermis was devoid of reaction product. Keratoacanthoma and the granular cells of verruca vulgaris were immunoreactive to the antibody for p21. Bowen's disease and squamous cell carcinoma were positive for p21, but basal cell carcinoma and seborrheic keratosis were negative. In mammary and extramammary Paget's diseases, the immunoreactivity was inconsistent. The expression of p21 in malignant melanoma cells was intense, whereas normal melanocytes and nevus cells were devoid of the expression. These results suggest that the expression of p21 does not correlate with nuclear anaplasia and malignant behaviour of cutaneous tumours.

Correlation between DNA quantities in agarose gel and chromatographic areas of photographed DNA bands.

To quantitate DNA from photographs, correlations between chromato-scanned (in the fluorescence mode) and virtual areas from white DNA bands on photographs were examined. A linear correlation between the two measurements with coefficients higher than 0.89 was observed. The dynamic range of the fluorescence detection yielded good linearity for a wide range of amounts of DNA (microgram-ng) on the photograph.

Genetic study on ossification of posterior longitudinal ligament.

Family studies and blood grouping tests were conducted in an attempt to elucidate the etiology and genetic mechanism of ossification of posterior longitudinal ligament of the spine (OPLL). Analysis of the collected date revealed the following results: Testing of inheritance pattern Fitness of OPLL to the hypotheses of simple recessive inheritance, simple dominant inheritance and multifactorial inheritance was tested, but fitness to any one of these hypotheses was statistically denied. Analyses of OPLL and genetic markers A comparative study was made between the OPLL patients and healthy donors residing in Tokyo on the phenotype frequency of the blood groups (eight systems), serum groups (six systems) and red cell enzyme groups (three systems). A significant association of OPLL with MN blood groups, Hp types and PGM1 types was observed. Examination of the correlation after combining MN and Hp showed the coefficient of contingency to be C = 0.6739 and that after the multiple combination of MN, Hp and PGM1 revealed the coefficient of contingency to be C = 0.8923, indicating a remarkably high correlation.

Multiple keratoacanthomas with peculiar manifestations and course.

The case of a 60-year-old Japanese man with unique multiple keratoacanthomas is reported. The lesions were located in the perioral area, grew peripherally, and healed centrally. Although individual lesions spontaneously regressed, prompt recurrence was observed after excision. Biopsy specimens taken from early proliferative, fully developed, and regressing stages revealed keratoacanthoma. Involucrin was demonstrated in the lesions during development and resolution. The early stage showed homogeneous staining of moderate intensity in the tumor cells at the suprabasal layers, and the staining became slightly irregular and intense when the lesions were fully developed. During resolution the intensity was markedly diminished. These results suggest that preterminal squamous differentiation accelerated during development of the tumors.

Alterations in membrane fluidity during keratinocyte differentiation measured by fluorescence polarization.

The epidermis shows a distinctive pattern of differentiation wherein keratinocytes proliferate in the basal cell layer and mature into spinous and granular cells. Using a discontinuous density-gradient centrifugation method, guinea-pig keratinocytes were separated into high (HDF), intermediate (IDF), and low (LDF) density fractions. Morphological and flow cytometrical observations demonstrated that HDF, IDF, and LDF were basal, spinous, and granular cell-rich fractions, respectively. Membrane fluidity of the fractionated keratinocytes was measured by diphenylhexatriene fluorescence polarization. Polarization (p)-value of keratinocytes was negatively correlated with temperature. At each temperature, HDF cells showed a lower p-value than IDF or HDF cells except at 40 degrees C. Since a low p-value indicates a high degree of Brownian motion, membrane fluidity is higher in basal cells and lower in spinous and granular cells. Our results indicate that membrane fluidity of guinea-pig keratinocytes decreases during their maturation.