ABSTRACT
Laser microdissection (LMD) with subsequent reverse transcription-PCR analysis is a powerful histochemical technique subserving the molecular characterization of specific cell types. We developed an efficient method for selective sampling of specific cell populations using immunohistochemistry coupled with LMD. The cerebral cortex of adult rats was cut into serial thin sections. Some sections were immunostained for parvalbumin. The adjacent sections were mounted on Cell Support Film for LMD and stained with neutral red. By comparison of the two adjacent sections, neuronal profiles representing parts of parvalbumin-immunopositive somata were identified in the neutral red-stained sections. These neuronal profiles were safely captured with LMD and analyzed on reverse transcription-PCR using extracted RNA. The method presented here can be applied to cell-type-specific characterizations using fixed cells under RNase-free conditions.
Subject(s)
Cerebral Cortex/chemistry , Histocytological Preparation Techniques , Immunohistochemistry/methods , Microdissection/methods , Neurons/chemistry , RNA, Messenger/analysis , Animals , Cerebral Cortex/cytology , Frozen Sections , Lasers , Male , Micromanipulation , Parvalbumins/analysis , Polymerase Chain Reaction/methods , Rats , Rats, Sprague-DawleyABSTRACT
An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.
Subject(s)
Central Nervous System/metabolism , Gene Expression Regulation , Ion Channels/biosynthesis , Ion Channels/genetics , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Aged , Aged, 80 and over , Alternative Splicing , Amino Acid Sequence , Animals , Blotting, Southern , Blotting, Western , Brain/metabolism , Cattle , Caudate Nucleus/metabolism , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , Cysteine Loop Ligand-Gated Ion Channel Receptors , Cytoplasm/metabolism , DNA, Complementary/metabolism , Dogs , Exons , Genes, Reporter , Green Fluorescent Proteins/metabolism , Hippocampus/metabolism , Humans , Introns , Ions , Kidney/metabolism , Male , Middle Aged , Molecular Sequence Data , Muscle, Skeletal/metabolism , Opossums , Pan troglodytes , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Polymerase Chain Reaction , Protein Sorting Signals , Protein Structure, Tertiary , RNA, Messenger/metabolism , Receptors, Serotonin/chemistry , Receptors, Serotonin/physiology , Recombinant Fusion Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue Distribution , TransfectionABSTRACT
Platelet-activating factor (PAF) has been reported to play a role in allergy and inflammatory reactions but its role in the pathogenesis of nasal polyps remains unclear. In this study, we examined both PAF and peptide leukotrienes (peptLTs) in individual preparations from nasal polyps. The amounts of PAF were much greater than those of peptLTs in all preparations. Nasal polyps were divided into two groups according to the severity of eosinophil infiltration: a severe group (eosinophil count > or = 50/mm2) and a mild group (eosinophil count < 50/mm2). The amounts of PAF in the nasal polyps were significantly higher in the severe group than in the mild group (p < 0.01). PAF activity correlated with tissue eosinophilia and polyps obtained from patients with aspirin-sensitive asthma contained relatively large amounts of PAF, with enriched infiltration of eosinophils.
