Kiyofumi Yamanishi

Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior

KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimers disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.

Low incidence of Ha-ras oncogene mutations in human epidermal tumors.

Activation of the Ha-ras oncogene by point mutations has been suggested to play a role in animal skin carcinogenesis models. In this study, we investigated the significance of the Ha-ras mutations in human epidermal tumors. DNAs from paraffin-embedded tissues of benign and malignant human epidermal tumors (27 samples from 25 patients) were prepared and examined for point mutations of codons 12, 13 and 61 of Ha-ras gene by polymerase chain reaction and oligonucleotide hybridization. Only one sample of basal cell carcinoma and one sample of keratoacanthoma were found to carry an A to T transversion at the second position of codon 61. This low incidence of Ha-ras mutations suggests that the mutational activation of the gene may not be primarily involved in human epidermal tumorigenesis.

Molecular cloning of human epidermal transglutaminase cDNA from keratinocytes in culture.

We have isolated a cDNA encoding human epidermal transglutaminase, a key enzyme of terminal differentiation of keratinocytes. A cDNA library from cultured human keratinocytes was screened by a PCR-amplified partial cDNA fragment of the enzyme with oligonucleotide primers based on the homology of the transglutaminase family. The cDNA is 2734 bp coding a protein of 817 amino acids. The several regions including the active site cysteine residue are highly conserved among the transglutaminase family. However, the charged N-terminal domain is unique to the epidermal transglutaminse, suggesting that the region is involved in the function of the enzyme in keratinocytes.

Cutaneous Metastasis from Hepatocellular Carcinoma Resembling Granuloma Teleangiectaticum

We reported a case of cutaneous metastasis originated from hepatocellular carcinoma, which appeared as a nodule resembling granuloma teleangiectaticum. The nodule, which was composed of tumor cells and intervening capillaries, showed characteristics similar to those of primary hepatocellular carcinoma. On immunohistochemical staining, the tumor cells were positive not only for AFP (alpha‐fetoprotein), but also for CEA (carcinoembryonic antigen), which is negative in most cases of primary hepatocellular carcinoma. We discussed this case with a review of previous reports of skin metastasis from liver carcinoma.

Possible role of calmodulin in stimulation of hexose transport by 12-O-tetradecanoylphorbol-13-acetate, a tumor promoter.

12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter stimulated the rate of 2-deoxy-D-glucose (2DG) transport. Three different kinds of calmodulin antagonists inhibited the TPA-stimulated 2DG transport, the mechanism of which was examined in kinetic analysis. These results indicate that the expression of the effect of TPA may depend on Ca2+-calmodulin system.

Ca2+-dependent stimulation of hexose transport by A23187, 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor in mouse fibroblasts

Ca2+ ionophore A23187 stimulated 2-deoxy-D-glucose (2DG) uptake in Swiss 3T3 mouse fibroblasts. Chelation of extracellular Ca2+ with ethylene-glycol-bis-(beta-aminoethylether) N,N-tetraacetic acid (EGTA) inhibited the effect of A23187. Similarly, the stimulation of 2DG uptake by a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was prevented by EGTA, whereas the epidermal growth factor (EGF)-stimulated 2DG uptake was not affected by EGTA alone, but in the presence of both EGTA and A23187 which effectively depleted cellular Ca2+ content, EGF could no longer stimulate 2DG uptake. These results suggest that Ca2+ regulates hexose transport system in Swiss 3T3 mouse fibroblasts, the activation of which by TPA and EGF differently depends on Ca2+.

Gene-knockout mice with abnormal epidermal and hair follicular development.

In the past 8 years, analysis of mutant mice and development of gene-knockout mice have provided important new avenues to identify disease genes and to study gene functions in the skin. Targeted disruption of genes in mice is a powerful means to investigate the contribution of a particular gene defect to a given phenotype and to generate murine models of hereditary skin disorders with epidermal and hair follicular abnormalities. This review summarizes recent studies of knockout mouse models with abnormalities in epidermal and/or hair follicular development.

Analysis of p53 gene mutations and loss of heterozygosity for loci on chromosome 9q in basal cell carcinoma.

To determine the role of the p53 gene in the pathogenesis of basal cell carcinoma (BCC), we screened mutations of the gene in 11 cases of BCCs using the polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP). However, in all the coding exons of the gene analysed, no evidence suggesting the mutations were obtained. On the other hand, in 2 of 5 informative cases of our BCCs (40%) we found loss of heterozygosity (LOH) for loci on chromosome 9q31 which is linked to the Gorlin syndrome, that predisposes to BCC. Therefore, we suggest that a putative tumor suppressor gene on the region of 9q, but not p53 gene, plays a critical role in the pathogenesis of BCC, independent of race.

Upstream activation element of the PH03 gene encoding for thiamine-repressible acid phosphatase in Saccharomyces cerevisiae

The PH03 gene of Saccharomyces cerevisiae encodes thiamine‐repressible acid phosphatase and requires the positively acting regulatory protein TH12 for its expression. Deletion analysis of the 5′‐flanking region of PH03 gene revealed that an activating region located at nucleotide position −234 to −215 relative to the translation initiation codon is required for the expression and sensitivity to thiamine. A chemically synthesized DNA fragment covering −234 to −215 showed a significant level of expression when inserted in front of the PH03 promoter lacking the activating region. Electrophoretic mobility shift assay demonstrated the presence of proteins that bound to the above DNA fragment in the nuclear extract from cells grown in thiamine‐free medium. These findings suggested that this region between −234 and −215 acts as an upstream activation element of the PH03 gene that can interact with regulatory proteins.

Differential detection of human papillomavirus DNA type 6 and 11 amplified by polymerase chain reaction

We have developed a method for differential detection of human papillomavirus type 6 and 11 with selective amplification of a segment of the viral DNAs by the polymerase chain reaction. The target sequence for amplification was a 134-bp (bp 206-339) in the E6 open reading frame of HPV 6 and 11 DNAs. DNA extracted from a paraffin-section or a minute fresh biopsy specimen of condyloma acuminatum was amplified and hybridized with an oligonucleotide probe specific for HPV 6 or 11 DNA. The method was applied to analyse 5 cases of genital condyloma acuminatum, and as a result, HPV 6 and 11 DNAs were detected in two and one case, respectively. This method is useful for differential diagnosis of HPV 6 and 11 infection.