Kanato Yamagata

Induction by lipopolysaccharide of cyclooxygenase-2 mRNA in rat brain; its possible role in the febrile response

Cyclooxygenase 2 (COX-2) is a newly discovered isoform of cyclooxygenase that is inducible by lipopolysaccharide (LPS) or cytokines. This enzyme is considered to play a major role in inflammatory processes by catalyzing the production of prostaglandins. In the present study, induction of COX-2 mRNA in the rat brain by intraperitoneal injection of LPS was studied by the in situ hybridization technique with special attention paid to timing and sites of induction along with the time course of fever. In situ hybridization was carried out on sections of rat brain, 1 h (latent phase), 2.5 h (maximally febrile phase), 4 h (plateau phase), and 7 h (recovery phase) after the LPS injection, as well as on those from the brains of untreated and saline-injected rats. Injection of LPS induced COX-2 mRNA in the brain in two different constituents: neuronal cells and non-parenchymal cells of the blood vessels and leptomeninges. Induction in the neuronal cells was restricted to some telencephalic areas where the COX-2 mRNA signal was also detected in control animals. The signal was maximally enhanced by 50 to 80% over the basal level 1 h after LPS injection. The COX-2 mRNA signal was hardly detectable in neuronal and glial cells in other brain regions, including the preoptic area, either in control or LPS-injected rats. Strong COX-2 mRNA signals, however, appeared in the inner surface of blood vessels and the leptomeninges over the entire brain, including the preoptic area and its vicinity. The signals were not detectable in the brains of control rats and were most intense in the brains of rats treated with LPS for 2.5 h or 4 h. These results demonstrate that two major cell groups in the brain, neuronal cells and non-parenchymal cells, are responsible for the enhanced production of prostaglandins after systemic LPS treatment. Considering the site and timing of induction, we propose a possible role for blood vessels and leptomeninges as the source of prostaglandin E2 in the genesis of fever.

Visinin: A novel calcium binding protein expressed in retinal cone cells

Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1). To investigate its function, visinin cDNA was isolated from a chick retinal lambda gt11 cDNA library, using anti-visinin serum. The beta-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody. The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells. Visinin mRNA was expressed only in the retinal photoreceptor layer. The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a Ca2+ binding protein. Visinin protein expressed in E. coli exhibited Ca2+ binding activity. These results suggest that visinin is a photoreceptor-specific Ca2+ binding protein and may be involved in phototransduction in the cone cells.

Induction of cyclooxygenase-2 mRNA in gerbil hippocampal neurons after transient forebrain ischemia

We examined the effect of brain ischemia on neuronal expression of cyclooxygenase-2 gene in the hippocampus. Transient forebrain ischemia was produced by occluding bilateral carotid arteries for 5 min in Mongolian gerbil. Northern blotting and in situ hybridization demonstrated that expression of cyclooxygenase-2 mRNA was transiently induced in the hippocampal neurons. Although future studies will be needed to clarify if induced cyclooxygenase-2 following ischemia is involved in neuronal damage or neuronal protection, selective cyclooxygenase-2 inhibitors may be a new therapeutical approach for the treatment of stroke.

Molecular Cloning and Characterization of Amida, a Novel Protein Which Interacts with a Neuron-specific Immediate Early Gene Product Arc, Contains Novel Nuclear Localization Signals, and Causes Cell Death in Cultured Cells

Amida was isolated by the yeast two-hybrid system as a novel protein which associated with Arc, a non-transcriptional immediate early gene specific to the brain. Amida was confirmed to be associated with Arc in vitro and in vivo. Amida shows no homology to known proteins. Amida is ubiquitously expressed, although it is abundant in the brain. A transfection study revealed that Amida was localized in the nucleus and after 72 h the transfected cells underwent apoptosis. Furthermore, we found two nuclear localization signals and a domain needed for interacting with Arc was encompassed by two nuclear localization signals. Co-transfection experiment with Amida and Arc suggested that Amida transported Arc into the nucleus and negatively regulated Amida-induced cell death. These results indicate that Arc together with Amida may modulate cell death in the brain.

Identification of glucocorticoid responsive elements (GREs) at far upstream of rat NPY gene

The location of three glucocorticoid responsive elements (GREs) in rat neuropeptide Y (NPY) gene was determined by chloramphenicol acetyltransferase (CAT) assay and nucleotide sequencing. We have reported that mRNA content of rat prepro-NPY is increased by 1.7-fold in NG108-15 cells by 1 microM dexamethasone, suggesting the presence of GRE in the gene. To identify the element, the 5-flanking DNA of 3.3 kilobases (kb) was isolated from rat NPY gene. When chimeric chloramphenicol CAT plasmids containing various deletions of the NPY upstream sequence were transfected into NG108-15 cells, the region between -2.9 and -2.1 kb relative to the cap site was found to potentiate the transcription of CAT gene in the presence of 1 microM dexamethasone. The nucleotide sequencing of this region revealed three GRE consensus sequences at -2.5, -2.2 and -2.1 kb. The results indicate that these elements present in the far upstream region of the NPY gene confer induction by glucocorticoids.

Multiple opsin mRNA species in bovine retina

Two retina specific cDNAs have been isolated by differential colony hybridization to retina and brain, and one of them, pCR-394, was identified as an opsin cDNA. By Northern hybridization experiment, the opsin cDNA hybridized to two species of bovine mRNA, one approximately 18 S (1800 bp) and the other 22 S (2600 bp). Using pCR-394 as a probe two opsin clones, R-5 (about 1200 bp) and LR-8 (about 2500 bp), were isolated from a cDNA library which was prepared by the method of Okayama-Berg. Each had a different length of 3-untranslated DNA. The nucleotide sequences of R-5 and LR-8, as well as Northern and Southern hybridization experiments suggest that at least two species of opsin mRNA are expressed from a single gene. When the effects of illumination were examined by Northern hybridization and translation assays, the ratio of the two opsin mRNA species was changed between light- and dark-adapted eyes.

Developmental changes of MEKA protein and opsin in normal and rd mice

Antisera raised against photoreceptor-specific MEKA and opsin proteins were provided for immunohistochemical studies on the retinas of normal (BJ57BL/6) and retinal degeneration (rd, C3H/He) mice. The expression of MEKA protein began at postnatal day 6 (P6) in the photoreceptor cells and gradually increased until P10 in both normal and rd mice. Thereafter the MEKA proteins in the photoreceptor cells of rd mice gradually decreased and disappeared at P18, whereas those of normal mice were increasing until P18. The time course of MEKA proteins in the photoreceptor cells of normal and rd mice was almost similar to that of opsin protein, except that the MEKA protein disappeared earlier than opsin.

Direct photosensitivity of chick pinealocytes as demonstrated by visinin immunoreactivity

SummaryVisinin, a calcium-binding protein isolated from the soluble fraction of homogenized chick retinae, has been recognized immunocytochemically in the pinealocytes of various submammals. In the chick pineal organ, continuous environmental light induced an increase in population density of visinin-immunoreactive pinealocytes. In semi-quantitative, dot-immunoblotting analysis, the amount of visinin in the pineal organs of chicks kept under continuous light for 3 days was 4–8 fold more abundant than that under continuous darkness for the same duration. Eye-enucleation and organ culture experiments clarified that this lighting effect was exerted directly on the pineal organ through the skull, and not via the neural pathway including the retinohypothalamic projection. These data suggest the existence of direct photosensitivity in the chick pinealocyte itself and the possible involvement of visinin in photoreception of the pineal organ as well as the retina of chicks.

Characterization and partial purification of a neurotrophic factor for cerebrocortical neurons from chicken cerebral extract

The survival of cerebrocortical neurons from 6 to 8-day-old chick embryos was investigated in a serum-free hormone-supplemented medium. The addition of cerebral extract promoted the survival of cortical neurons in a dose-dependent manner, but induced almost no neurite outgrowth. The trophic activity was higher in the adult cerebrum than in the embryonic cerebrum. The tropic factor was partially purified from adult chicken cerebrum, and the molecular weight of the factor was estimated to be about 60 kDa. The activity for survival factor was fairly resistant to heat or trypsin treatment. When the partially purified sample was, however, treated with trypsin (1 mg/ml) for 20 h and applied to a TSK G2000 SW gel filtration column, the activity moved from 60 to 70 kDa untreated active fractions to fractions with about 10 kDa. These physicochemical properties of the survival factor suggest a new class of macromolecular trophic factors in the brain.