Yasutsugu Nakashima

Comparative Studies of the Detection Rates of Leishmania Parasites from Formalin, Ethanol‐fixed, Frozen Human Skin Specimens by Polymerase Chain Reaction and Southern Blotting

In this study, detection rates of Leishmania parasites from human skin were compared among three different types of specimens, formalin‐fixed, ethanol‐fixed, and frozen, by polymerase chain reaction (PCR) and Southern blotting. For this purpose, we used biopsy specimens collected from 19 leishmaniasis patients and performed PCR and Southern hybridization with the probe specific for Leishmania (Viannia) braziliensis complex. Among these 19, 16 specimens were from cutaneous leishmaniasis (CL), one, diffuse cutaneous leishmaniasis (DCL) and 2, mucocutaneous leishmaniasis (MCL) and were formalin‐fixed and paraffin‐embedded. The causative agents for one case of CL and one case of DCL were already identified as L. (Leishmania) complex. Six specimens of CL were preserved in 100% ethanol. Two specimens of MCL were frozen tissues. PCR using the formalin‐fixed and paraffin‐embedded specimens revealed positive bands at 70bp in 9 (47.4%) out of 19 specimens of CL, MCL and DCL. Southern blotting detected the signals in 12 (63.2%) out of the 19. PCR using the 100% ethanol‐fixed specimens revealed positive bands in 4 (66.7%) out of 6, and Southern blotting also detected the signals in 4 (66.7%) out of the 6. PCR and Southern blotting using 2 frozen specimens of MCL were always positive (100%). Although we failed to detect significant differences by Chi‐square test between the results from the formalin‐fixed, paraffin‐embedded specimens and those from 100% ethanol‐fixed ones, we concluded that ethanol‐fixed specimens, convenient for transportation and storage, would be more useful for diagnosis of leishmaniasis by PCR in a developing country.

Association of human papillomavirus type 16 with malignant melanoma

We report a case of malignant melanoma associated with human papillomavirus (HPV) in a 37-year-old woman. The patient has had numerous brown papular and nodular tumors, 5 to 30 mm in diameter, on her left leg for > 15 years, some of them coalescing rapidly in the last 12 months to a multilobulated black nodule diagnosed as malignant melanoma by histology and immunohistochemistry. HPV type 16 DNA was detected in the melanoma specimen by reverse transcriptase polymerase chain reaction (rt-PCR) and in situ hybridization (ISH) of the tumor tissues. This is the first report of melanoma associated with HPV 16.

Alteration of mRNA levels of δ-aminolevulinic acid synthase, ferrochelatase and heme oxygenase-1 in griseofulvin induced protoporphyria mice

Human erythropoietic protoporphyria (EPP) is an inherited disorder of porphyrin metabolism and its experimental murine model can be produced by treatment with griseofulvin (GF). We investigated the alteration of mRNA expression in ferrochelatase (FeC), delta-aminolevulinic acid synthase (ALAS) and heme oxygenase-1 (HO-1) in liver, skin and peripheral blood cells of GF-treated mice. In liver, ALAS mRNA was enhanced dramatically by GF administration, in accord with thesis that the expression of ALAS is regulated by feedback mechanism. The expression of HO-1 mRNA increased most rapidly and drastically in liver, however its mechanism of regulation may be different from that of ALAS mRNA. The level of FeC mRNA in liver was less affected with GF treatment. Our results indicate that the inhibition of FeC by GF administration might occur primarily at post-transcriptional level. Similar effects were observed in the ALAS and HO-1 mRNA expression in peripheral blood cells, 2-fold increase in the ALAS mRNA and increase from undetectable level to detectable level in the HO-1 mRNA. In skin of GF-treated mice, average increases of 1.3-fold in the ALAS mRNA and 1.6-fold in the HO-1 mRNA were statistically insignificant. The FeC mRNA level was not altered in peripheral blood or in skin of GF-treated mice. The present study indicates that the molecular analysis is practicable in skin and peripheral blood. In further study, this model could contribute to investigate the pathogenesis of clinical manifestation including possibly cutaneous changes in EPP.

Quantitative analysis of ferrochelatase mRNA in blood cells of erythropoietic protoporphyria patients

Ferrochelatase (FC; heme synthetase, EC 4.99.1.1.) catalyses the synthesis of heme from protoporphyrin IX, the final step in the heme synthetic pathway. The hereditary deficiency of this enzyme gives rise to erythropoietic protoporphyria (EPP). We developed a rapid, non-radioactive means of measuring human FC mRNA levels in the EPP patients. It is based on the reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the RNA obtained from peripheral blood. The amplified DNA was detected by agarose gel electrophoresis with ethidium bromide staining and the fluorescent intensity was measured by scanning densitometry applied directly to Polaroid 665 negative film. The relative expression level of FC mRNA, compared with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, was estimated at several points in the exponential phase of PCR cycles or at a point in the exponential phase of PCR performed on serially diluted the cDNA samples. The estimate of the FC mRNA by this method correlated well with the level of the FC mRNA measured by Northern blotting in the EB virus-transformed lymphocytes of the same patients. The level of the FC mRNA appeared to vary among the patients in whom a decreased level of enzymatic activity was indicated.

Expression of PDGF and C‐myc in Atherosclerotic Lesions in Cholesterol‐Fed Chicken

Platelet-derived growth factor (PDGF) has been reported to be a potent mitogen for mesenchymal cells such as smooth muscle cells and glial ce1ls.l Several reports2,’ have indicated that protooncogenes such as c-myc and c-fos may be involved in the mechanisms of atherosclerosis. The aim of this study is to investigate the role of PDGF and c-myc on the development of atherosclerotic lesions in chickens, which serve as a useful experimental model for the study of athero~clerosis.~ Experimental animals consisted of two groups. Six 4-month-old roosters were fed an atherogenic diet that contained 2% cholesterol and 10% corn oil for 3 months. Five age-matched normal roosters were fed basal diet. The thoracic and abdominal aortas were collected and histologically examined. Immunostaining was performed by the avidin-biotin-peroxidase complex (ABC) (Vector Labs., Burlingame, Calif.) method5 using primary antibodies of PDGF-A, PDGF-B (genzyme), PDGF receptor, Pan-myc (Cambridge Research Biochemicals Ltd.). In situ hybridization was carried out according to the method of Coxet ~ 1 . ~ Probes for detecting the PDGF-A, PDGF-B, PDGF-A receptor, and PDGF-B receptor mRNAs were purchased from Oncogene Science, Inc., and were biotinylated with the terminal labeling kit (Enzo Biochem, Inc.). The probe for c-myc mRNA was prepared by inserting the Sma I cut fragment (exon 11) of pMyc6.514 (offered by Japanese Cancer Research Resources Bank) into plasmid pSP65. According to the modified method of Melton et al.,’ biotinylated RNA probes were synthesized in vitro using sP6 RNA polymerase, the linearized plasmid DNA, and Biotin 1 I-UTP. Biotinylated probes on slides were visualized with Blu GENEm (BRL). A lipid-rich thickened intimal lesion was more frequently seen in the thoracic aortas than in the abdominal aortas of cholesterol-fed roosters. Immunohistochemicat examination disclosed no significant expression of PDGF-A, -B, PDGF receptors, and c-myc in the entire aorta of normal roosters. In cholesterol-fed roostcrs, the intense reaction of PDGF-B, PDGF receptor (PDGF-R), and c-myc was seen in lipid-rich thickened intimal lesions of the entire aorta while no significant reaction of PDGF-A was observed in the same lesions. In situ hybridization study demonstrated

Expression of platelet-derived growth factor and c-myc in atherosclerotic lesions in cholesterol-fed chickens: immunohistochemical and in situ hybridization study

Immunohistochemical examination showed no significant expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF receptors, or of c-myc in the thoracic and abdominal aortas of normal roosters. In cholesterol-fed roosters, intense immunohistochemical reaction for PDGF-B, PDGF receptor, and c-myc was seen in the lipid-rich thickened intimal lesions of the thoracic and abdominal aortas while no significant immunoreaction for PDGF-A was demonstrated in the same lesions. In accordance with immunohistochemical findings, in situ hybridization demonstrated a significant level of expression of PDGF-B, PDGF-A receptor, PDGF-B receptor, and c-myc genes in proliferating intimal cells of the thoracic and abdominal aortas. These results suggest that coordinate actions of PDGF-B and c-myc play an important role in proliferation of intimal cells in the developing atherosclerotic lesions in chickens.