Establishment of an in vitro cell line experimental system for the study of inhalational anesthetic mechanisms.

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia.

Characterization of sevoflurane effects on Per2 expression using ex vivo bioluminescence imaging of the suprachiasmatic nucleus in transgenic rats.

The inhalation anesthetic sevoflurane suppresses Per2 expression in the suprachiasmatic nucleus (SCN) in rodents. Here, we investigated the intra-SCN regional specificity, time-dependency, and pharmacological basis of sevoflurane-effects. Bioluminescence image was taken from the SCN explants of mPer2 promoter-destabilized luciferase transgenic rats, and each small regions of interest (ROI) of the image was analyzed.

Rapid modulation of hypothalamic Kiss1 levels by the suckling stimulus in the lactating rat.

In mammals, lactation suppresses GnRH/LH secretion resulting in transient infertility. In rats, GnRH/LH secretion is rescued within 18-48 h after pup separation (PS) and rapidly re-suppressed by subsequent re-exposure of pups. To elucidate the mechanisms underlying these rapid modulations, changes in the expression of kisspeptin, a stimulator of GnRH secretion, in several lactating conditions (normal-lactating; 4-h PS; 18-h PS; 4-h PS +1-h re-exposure of pups; non-lactating) were examined using in situ hybridization.

Epigenetic suppression of mouse Per2 expression in the suprachiasmatic nucleus by the inhalational anesthetic, sevoflurane.

Background: We previously reported that sevoflurane anesthesia reversibly suppresses the expression of the clock gene, Period2 (Per2), in the mouse suprachiasmatic nucleus (SCN). However, the molecular mechanisms underlying this suppression remain unclear. In this study, we examined the possibility that sevoflurane suppresses Per2 expression via epigenetic modification of the Per2 promoter.

Chromosome-specific segmentation revealed by structural analysis of individually isolated chromosomes.

Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.

Amplification of the ABCB1 region accompanied by a short sequence of 200bp from chromosome 2 in lung cancer cells.

Lung cancer sublines No15-80-1 and No15-80-6 were selected by treatment of cell line NCI-H460 with paclitaxel at stepwise increasing concentrations from 50 nmol/L to 800 nmol/L. The two sublines exhibited amplifications of the ABCB1 region (previously MDR1) with different copy number profiles, but shared a common amplification pattern, which has been observed in amplification mediated by the breakage-fusion-bridge (BFB) cycle. Sequence analysis of the distal ends of the amplified regions, which were probably generated in a break-and-fusion of the initial round of the BFB cycle, revealed a head-to-head fused sequence of chromosome 7.

Expression of porcine FSHbeta subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic rats.

A transgenic rat was established using the construct of porcine FSHbeta subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHbeta was only expressed in the pituitary.

High level expression of Prop-1 gene in gonadotropic cell lines.

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism.

Transcriptional activity of the 5' upstream region of the porcine glycoprotein hormone alpha subunit gene.

Gene expression of the porcine glycoprotein hormone alpha subunit (p-alphaGSU) was examined in LbetaT2 cells, which were established from the anterior pituitary lobe of the immortalized transgenic mouse and produce alphaGSU, and in CHO cells cloned from Chinese hamster ovaries. Expression of the reporter gene fused with p-alphaGSU gene upstream in LbetaT2 cells showed that the distal regions -540/-240 and -798/-541 are important for the activation of gene expression. In contrast, the transcriptional activity of the distal region of p-alphaGSU gene was repressed in CHO cells.

Prominent expression of spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1 in LH-producing cells, LbetaT2.

The LH-producing cell line, LbetaT2, and non LH-producing cell line, alphaT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells.

Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2.