Hidetaka Nagata

In situ expression of corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) genes in human skin

Systemic stresses induce corticotropin‐releasing hormone (CRH) expression in hypothalamus. CRH is released to the pituitary gland, where it stimulates proopiomelanocortin (POMC) production acting via the CRH receptor (CRH‐R). CRH and POMC peptides are also detected in sites outside of the central nervous system (CNS), such as the skin. However, it has not been elucidated whether these peptides detected in the skin are derived from CNS or are produced locally. Using immunohistochemical and in situ reverse‐transcription (RT)‐PCR techniques, we demonstrated coexpression of CRH and POMC mRNAs in the epidermis and pilosebaceous units of the human skin. This coexpression was confirmed by the combination of laser‐capture microdissection (LCM) with RT‐PCR, analyzing mRNA expressions in captured sebaceous cells. Immunoreactivities and expressions of CRH and POMC mRNAs were strong in inflammatory lesions, melanocytic nevus, seborrheic keratosis, and also in the periphery of the benign tumor. These findings suggest that CRH and POMC peptides are produced locally in the skin and are regulated by inflammatory cells as well as by autocrine mechanisms. The skin may have “a local stress response system,” whose activity is mediated by CRH and POMC peptides, in an equivalent to hypothalamus‐pituitary adrenal axis.

Expression and localization of leptin receptor in the normal rat pituitary gland

Abstract. Leptin receptor (leptin-R) is a polypeptide consisting of a single transmembrane-spanning component. Recent studies performed by reverse transcriptase polymerase chain reaction (RT-PCR) have shown the production of leptin-R in various tissues including the pituitary, hypothalamus and reproductive organs. The localization of leptin-R protein in the pituitary gland, however, has not been extensively studied. This study deals with the expression of leptin-R in the normal rat pituitary gland, which was disclosed primarily in the plasma membrane fraction by immunoblotting and immunohistochemical staining methods. Double immunohistochemical staining revealed that the colocalization of leptin-R and anterior pituitary hormone expression was seen mainly in growth hormone (GH)-secreting cells (97.4±1.3%; GH-positive cells/leptin-R-positive cells), but in less than 1% of prolactin (PRL)-, adrenocorticotropic hormone (ACTH)-, thyroid-stimulating hormone-β (TSHβ)- and follicle-stimulating hormone-β (FSHβ)/luteinizing hormone-β (LHβ)-positive cells. In contrast, leptin was localized most frequently in FSHβ/LHβ- and less frequently in TSHβ-positive cells. The above findings suggest that, in the rat anterior pituitary gland, there are paracrine relationships between leptin-producing cells and cells with leptin-R, which may regulate the function of GH cells.

Differentiation of necrotic cell death with or without lysosomal activation: application of acute liver injury models induced by carbon tetrachloride (CCL4) and dimethylnitrosamine (DMN).

We investigated the relationship between DNA degradation and lysosome activity (loss of lysosomal integrity) in necrotic cell death induced by carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN): coagulation necrosis and hemorrhagic necrosis, respectively. TdT-mediated dUTP–biotin nick end-labeling (TUNEL) and enzyme histochemistry for acid phosphatase were performed in both models and results were analyzed by light microscopy, electron microscopy, and confocal laser scanning microscopy (CLSM). In the CCl4-injected liver, TUNEL staining was closely associated with release of lysosomal enzymes into the cytoplasm, and intranuclear deposition of lysosomal enzymes took place at an early stage of subcellular damage. In the DMN-injected liver, TUNEL-positive nuclei tended to have well-preserved lysosomes and centrally localized TUNEL signals. It was assumed that acute hepatocellular damage in the CCl4-injected liver would be characterized by necrotic cell death with lysosome activation and that damage in the DMN-injected liver would be necrotic cell death without lysosome activation. In the DMN-injected liver, DNA degradation may be selectively induced in the nuclear center, in which heterochromatin (including inactive chromatin) is believed to be a target. We concluded that necrotic cell death, i.e., DNA degradation, would be at least divided into two types, with/without association with lysosome activation, represented by necrotic cell death in the CCl4-injected liver and that in the DMN-injected liver.

Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC α and βII in Fetal Rat Cerebral Cortex Cultured Neuron

Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC α and PKC βII species.

Stimulation of melanogenesis by nordihydroguaiaretic Acid in human melanoma cells.

Nordihydroguaiaretic acid (NDGA), a lignan found in vegetables, fruits and legumin, has been shown to possess antineoplastic, antiviral and antioxidant characteristics. In this study, we examined the effect of NDGA on melanogenesis in human melanoma cells (HMVII). In vitro, NDGA does not alter mushroom tyrosinase activity. However, in NDGA-treated HMVII cells, cellular tyrosinase activity increased in both a time- and dose-dependent manner. The concomitant increases in melanin content in NDGA-treated cells indicated an elevation of melanin synthesis by tyrosinase activation. In addition, after a 7-day incubation, melanin content in 20 μM NDGA-treated cells increased 5.02 fold. Tyrosinase protein also increased by treatment with NDGA. Nevertheless, tyrosinase mRNA was not altered in NDGA-treated cells. Our results suggest that NDGA can increase tyrosinase activity and de novo synthesis of melanin in human melanoma cells. We found that NDGA is a novel potent stimulator of melanogenesis in human melanoma cells.