Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures

Tsuchiya, Remi; Yoshiki, Fumito; Kudo, Yoshihisa; Morita, Mitsuhiro

doi:10.1016/s0006-8993(02)03518-7

Cited by 55 publications

(31 citation statements)

References 39 publications

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“…Hippocampal primary culture and cortical astrocyte-enriched culture were carried out as described previously (18,19). In brief, the hippocampal cells were prepared from embryonic day 18 or 19 Wistar rats by enzymatic treatment and plated onto 12-mm polyethyleneimine-coated coverslips in DMEM containing 50 units/ml penicillin, 50 g/ml streptomycin, 5% horse serum, and 5% FBS.…”

Section: Isolation and Construction Of Mouse Plc2 Cdna-mentioning

confidence: 99%

A Novel Phospholipase C, PLCη2, Is a Neuron-specific Isozyme

Nakahara¹,

Shimozawa²,

Nakamura³

et al. 2005

Journal of Biological Chemistry

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116

113

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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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Section: Isolation and Construction Of Mouse Plc2 Cdna-mentioning

confidence: 99%

A Novel Phospholipase C, PLCη2, Is a Neuron-specific Isozyme

Nakahara¹,

Shimozawa²,

Nakamura³

et al. 2005

Journal of Biological Chemistry

Self Cite

116

113

View full text Add to dashboard Cite

show abstract

“…When the cultured cells reached 70-80% confluence, they were sub-cultured at a ratio of 1:2 using 0.25% (w/v) trypsin. To obtain the highest efficiency of transfection and low cytotoxicity, transfection conditions were optimized by varying the cell density and the concentrations of plasmid DNAs (Clontech) for pEGFP-N3-L-FABP and Lipofectamine 2000 (Invitrogen), according to the Lipofectamine medium methods of Escriou et al (2001) and Tsuchiya et al (2002). Control cells and cells transfected with pEGFP-N3-L-FABP were lysed for total protein extraction.…”

Section: Over-expression Of Recombinant L-fabp Gene In Hepatocytes Anmentioning

confidence: 99%

Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library

Gao

Zhang

et al. 2015

Genet. Mol. Res.

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ABSTRACT. Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

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“…The electrophoretic mobilities of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were determined using the polyacrylamide gel electrophoresis protocol described by Marvin (1977) of American Type Culture Collection. Using the method of Tsuchiya et al (2002), the fluorescent protein genes pEGFP-C1, pEGFP-N3, pEYFP-N1, and pDsRed1-N1 were transfected into cells with Lipofectamine 2000. The cells were observed at 24, 48, and 72 h after transfection using a laser confocal microscope.…”

Section: Biological Analysismentioning

confidence: 99%

Attempt at conserving the genetic resources of Hu sheep by fibroblast line cryopreservation

et al. 2013

Journal of Applied Animal Research

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Hu sheep is one of the most distinctive local sheep breeds in China, while investigations concerning its cell biology and molecular genetics are still scarce. To preserve the valuable genetic resources of the Hu sheep, we established a Hu sheep fibroblast line from 35 sheeps' ear marginal tissues using explant culture and cryopreservation techniques, and its biological characteristics were identified, including cell morphology, growth curve, karyotype, isoenzyme polymorphism, and expression of exogenous fluorescent genes. The results showed that the viability of the fibroblasts before freezing was 96.54 ± 3.22% and 92.36 ± 2.77% after thawing, and there was no microbial contamination and crosscontamination; the growth curve of Hu sheep ear marginal fibroblasts displayed an obvious "S" shape; all somatic chromosomes were acrocentric autosomes, and only the two sex chromosomes (XY) were submetacentric; the normal number of chromosomes (2n = 54) was observed in 96.56 ± 2.89% of the cells; and the transfer efficiencies of four fluorescent proteins could achieve 37.4% with an optimized ratio of plasmid and lipofectamine. It could be concluded that this study has not only provided the biological characteristics of Hu sheep at cellular level, but also makes a valuable contribution to the preservation of the genetic resources of the Hu sheep.

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Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures

Cited by 55 publications

References 39 publications

A Novel Phospholipase C, PLCη2, Is a Neuron-specific Isozyme

A Novel Phospholipase C, PLCη2, Is a Neuron-specific Isozyme

Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library

Attempt at conserving the genetic resources of Hu sheep by fibroblast line cryopreservation

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