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Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, ␤-, ␥-, ␦-, ⑀-, -, and -type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC2. PLC2 is composed of conserved domains including pleckstrin homology, EFhand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC2 was more sensitive to calcium concentration than the PLC activity of the PLC␦-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC2 in pyramidal cells of the hippocampus. Finally, we found that PLC2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.Phosphoinositide metabolism contributes an important intracellular signaling system that is involved in a variety of cell functions such as hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, and regulation of the cytoskeleton (1-4). Phospholipase C (PLC) 1 is a key enzyme in this system and acts by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate two second messengers, inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Diacylglycerol mediates the activation of protein kinase C, and IP 3 releases Ca 2ϩ from intracellular stores (5, 6). Twelve mammalian PLC isozymes have been cloned so far, and they are divided into six classes on the basis of structure and activation mechanisms: ␤(1-4)-, ␥(1, 2)-, ␦(1, 3, 4)-, ⑀(1)-, (1)-, and (1)-type (1, 3, 7-13). All of the PLC isozymes contain catalytic X and Y domains as well as various regulatory domains, including the protein kinase C conserved region 2 (C2) domain, EF-hand motif, and pleckstrin homology (PH) domain. Subtype-specific domains contribute to specific regulatory mechanisms. These domains include the Src homology domain in PLC␥ (3) and the Ras-associating domain and Ras-GTPase exchange factor-like domain in PLC⑀ (14, 15).Recent data base information suggests the existence of an additional PLC-like protein in the mouse and human genomes. Therefore we attempted to isolate the cDNA for PLC-like protein, PLC2, from mouse...

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A major obstacle in Alzheimer’s disease (AD) research is the lack of predictive and translatable animal models that reflect disease progression and drug efficacy. Transgenic mice overexpressing amyloid precursor protein (App) gene manifest non-physiological and ectopic expression of APP and its fragments in the brain, which is not observed in AD patients. The App knock-in mice circumvented some of these problems, but they do not exhibit tau pathology and neuronal death. We have generated a rat model, with three familiar App mutations and humanized Aβ sequence knocked into the rat App gene. Without altering the levels of full-length APP and other APP fragments, this model exhibits pathologies and disease progression resembling those in human patients: deposit of Aβ plaques in relevant brain regions, microglia activation and gliosis, progressive synaptic degeneration and AD-relevant cognitive deficits. Interestingly, we have observed tau pathology, neuronal apoptosis and necroptosis and brain atrophy, phenotypes rarely seen in other APP models. This App knock-in rat model may serve as a useful tool for AD research, identifying new drug targets and biomarkers, and testing therapeutics.

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), a counterpart of the human growth-regulated oncogene product (GRO), has been suggested to participate in neutrophil recruitment in an experimental model of gastritis in rat. However, the mechanism(s) involved in regulation of CINC-1 production by the gastric mucosa remains unclear. The aim of this study was to investigate the mechanism(s) of CINC-1 production by rat gastric mucosa in vitro. All experiments were performed using rat normal gastric mucosal cell line (RGM-1). RGM-1s were stimulated with tumor necrosis factor (TNF)-␣, and CINC-1 mRNA levels (reverse transcription-polymerase chain reaction) and protein secretion (enzyme-linked immunosorbent assay) were assessed. The production of reactive oxygen species (ROS) and nuclear factor (NF)-B activation (translocation to the nuclei) in response to TNF-␣ stimulation was evaluated using fluorescence microscopy in the presence or absence of the inhibitors of mitochondrial electron flow and NF-B activation. Stimulation of RGM-1 cells with TNF-␣ resulted in an increase in intracellular oxidative stress, NF-B translocation to the nuclei, and up-regulation of CINC-1 mRNA and protein, which was prevented by interfering with mitochondria-dependent ROS production and NF-B activation. Taken together, these findings indicate that CINC-1, a counterpart of the human GRO, production by rat gastric epithelial cells in response to TNF-␣ stimulation is an oxidant stress-mediated and NF-Bdependent event.