Minoru Oshiro

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing.

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.

Association of human papillomavirus type 6 with a verruciform xanthoma.

We report a case of verruciform xanthoma (VX) associated with human papillomavirus (HPV) in a 67-year-old male. The patient had a pale-reddish, granular and verrucous tumor on the right side of his scrotum for four years. Histopathologic examination showed typical features of VX. HPV was detected by immunohistochemistry, electron microscopy, and PCR examinations. Ultrastructural examination revealed virus-like particles of 40–50 nm in the nucleus of the upper epidermal keratinocytes. HPV type 6a DNA was detected in lesional tissue by polymerase chain reaction and sequence analysis. To the best of our knowledge, this is the first case report of VX associated with HPV.

MINK is a Rap2 effector for phosphorylation of the postsynaptic scaffold protein TANC1.

Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.

Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.

Rap2 function requires palmitoylation and recycling endosome localization

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.

Polymorphisms of cytochrome b gene in Leishmania parasites and their relation to types of cutaneous leishmaniasis lesions in Pakistan

The exact species and/or strains of Leishmania parasites involved strongly influence the clinical and epidemiological features of leishmaniasis, and current knowledge of those influences and relationships is inadequate. We report that cytochrome b (cyt b) gene sequencing identified causal Leishmania parasites of 69 cutaneous leishmaniasis cases in Pakistan over a 3‐year period. Of 21 cases in highland areas (Quetta city, Balochistan province), 16 (76.2%) were identified as Leishmania (L.) tropica and five (23.8%) as Leishmania (L.) major. Of 48 cases from lowland areas, cities/villages in Indus valley in Sindh and Balochistan provinces, 47 (97.9%) were identified as L. (L.) major and one (2.1%) as L. (L.) tropica. Statistical analysis (Fishers exact test) revealed a significant difference (P < 0.0001) in the distribution of the two species by altitude; L. (L.) major is predominant in lowland and L. (L.) tropica at highland areas. The present result enriched our earlier finding, based on the first years cultured parasite data, that only L. (L.) tropica was found in highland areas and only L. (L.) major in lowland areas. Among Leishmania samples analyzed, three types of cyt b polymorphism of L. (L.) major were found, including 45 (86.5%) cases of type I, six (11.5%) of type II and one (2%) of type III. We report for the first time on the presence of polymorphisms in L. (L.) major (types I, II and III) based on species identification using cyt b gene sequencing from clinical samples. Moreover, we found no correlation between clinical presentation (wet‐, dry‐ and/or mixed‐types of cutaneous lesions) and causal Leishmania parasites.

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.

Detection of human papilloma virus type 56 in extragenital Bowen's disease

A case of Bowens disease arising on the medial part of the first metatarsal bone of an 81-year-old Japanese woman is described. Histopathologically, proliferation of atypical cells was found throughout the epidermis. Electronmicroscopy revealed virus particles 40-50 nm in diameter in the nuclei of tumour cells at the granular cells just on or below the horny layer. Positive bands were obtained by polymerase chain reaction using a consensus primer of human papilloma virus L1 portion. Sequencing analysis of the amplified DNA revealed the same base sequences and homology as human papilloma virus 56. To the best of our knowledge, this case is the first report in which human papilloma virus 56 was found in a case of extragenital Bowens disease. We consider it important to understand that human papilloma virus 56, often found in cervical lesions, can be detected in extragenital Bowens diseases.

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.

An imported case of cutaneous leishmaniasis caused by Leishmania (Leishmania) donovani in Japan

Leishmaniasis is a major world health problem, and 12 million people are estimated to be infected in 88 countries. There have been few reports of leishmaniasis in Japan and all were of foreign origin; therefore diagnosis is difficult for Japanese physicians. There are 21 different pathogenic Leishmania species, and identification is obtained by polymerase chain reaction (PCR). Here we report an imported case of leishmaniasis by Leishmania (Leishmania) donovani infection from Sri Lanka. L. (L.) donovani usually causes visceral leishmaniasis, but in this case, the patient manifested cutaneous leishmaniasis. The identification of Leishmania species by PCR and investigation of the patients background such as nationality and disease endemicity are important for diagnosis and treatment. This is the first report of cutaneous leishmaniasis by L. (L.) donovani in Japan.