Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.     

Injection of the insulin receptor alpha subunit increases blood glucose levels in mice

Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.     

The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: possible role of placenta-specific basic helix-loop-helix transcription factor, human achaete-scute homologue-2

PACE4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) beta family proteins. We previously reported that PACE4 is highly expressed in syncytiotrophoblasts of human placenta [Tsuji et al. (2003) BIOCHIM: Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The overexpression of Hash-2 caused a marked decrease in PACE4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE4 increased. In both cases, other SPCs, such as furin, PC5/6, and PC7/8, were not affected. Further, PACE4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotrophoblast layer where PACE4 was not expressed. In contrast, the PACE4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE4 activity.     

Most apoptotic cells in mdx diaphragm muscle contain accumulated lipofuscin

An age-related pigment, lipofuscin (LF), which accumulates in postmitotic, long-lived cells, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals. In the present study we show that LF is accumulated in some myofibres, myosatellite cells and interstitial cells in the diaphragm muscles of the X chromosome-linked muscular dystrophic (mdx) mice at the age of 10 weeks when repetitive cycles of de- and regeneration of myofibres occur. In contrast, LF is virtually absent in diaphragm muscles of age-matched C57BL/10 (C57) normal control mice. Therefore, mdx muscle is more susceptible to oxidative stress than normal muscle. We hypothesise that gene-regulated cell death (apoptosis) occurs in dystrophic muscle cells that accumulate LF as a consequence of either oxidative stress or injury. We found that 74-79% of apoptotic myosatellite cells, interstitial cells and myofibres in mdx diaphragm contain accumulated or dotted LF granules, but only 12-20% of non-apoptotic cells contain LF. Apoptotic cells are very rare in the diaphragm of age-matched C57 control mice. This suggests that the regeneration of mdx diaphragm muscle initiated from myosatellite cells is impaired by their apoptosis as the result of either oxidative stress or a product of oxidative injury.     

Phylogenetic position of Eulipotyphla inferred from the cDNA sequences of pepsinogens A and C

Although to date the phylogenetic position of the provisional order Eulipotyphla has been assessed by various molecular markers, it has not been conclusively clarified due to low statistical supporting values and inconsistent results. To clarify the phylogenetic position of Eulipotyphla, we cloned cDNAs for pepsinogens A and C from five mammalian species belonging to four different orders and determined their nucleotide sequences. Molecular phylogenetic analysis based on the 1st and 2nd codon positions of the protein-coding region of cDNA sequences strongly supported the close relationship between Eulipotyphla and Chiroptera. Carnivora was found to be a sister group to these two orders. The monophyly of the order Rodentia and that of the cohort Glires (Rodentia and Lagomorpha) was also shown by the present phylogenetic trees of pepsinogens.     

Base mutation analysis by a ferrocenyl naphthalene diimide derivative

An electrochemical DNA sensing technique was developed by using ferrocenyl naphthalene diimide coupled with a probe DNA-immobilized electrode. This technique enabled detection of target DNA quickly and with high sensitivity. Applicability of this technique for single nucleotide polymorphisms (SNPs) analysis was demonstrated by successful analysis of a DNA mismatch on the cancer repression gene p53 with high precision.     

Neurotoxic mechanisms triggered by Alzheimer's disease-linked mutant M146L presenilin 1: involvement of NO synthase via a novel pertussis toxin target

While it has been reported that familial Alzheimer's disease (FAD)-linked mutants of amyloid precursor protein (APP) and presenilin (PS)2 induce neuronal cytotoxicity in a manner sensitive to antioxidant and pertussis toxin (PTX), little of the mechanism for PS1-mediated neuronal cell death has been characterized. We previously found that multiple mechanisms, different in detail, underlie cytotoxicities by two FAD-linked mutants of APP, using neuronal cells with an ecdysone-controlled expression system. Here we report that this system revealed that (i) low expression of FAD-linked M146L-PS1 caused neuronal cell death, whereas that of wild-type (wt)PS1 did not; (ii) mutation-specific cytotoxicity by M146L-PS1 was sensitive to antioxidant glutathione-ethyl-ester and resistant to Ac-DEVD-CHO; (iii) cytotoxicity by higher expression of wtPS1 was resistant to both; and (iv) cytotoxicity by M146L-PS1 was inhibited by PTX. It was also highly likely that the involved superoxide-generating enzyme was nitric oxide synthase (NOS), and that the PTX-sensitive cytotoxic signal by M146L-PS1 was mediated by none of the G(i/o) proteins. We conclude that M146L-PS1 activates a NOS-mediated cytotoxic pathway via a novel PTX target.