A domino pericyclic route to polysubstituted salicylic acid derivatives: four sequential processes from enynones and ketene silyl acetals.

Alkenyl alkynyl ketones and ketene silyl acetals (KSAs) undergo regioselective [2+2]-cycloaddition under thermal conditions, triggering domino pericyclic reactions en route to various poly-substituted salicylic acid derivatives.

Siloxy(trialkoxy)ethene undergoes regioselective [2+2] cycloaddition to ynones and ynoates en route to functionalized cyclobutenediones.

Regioselective [2+2] cycloaddition of ynones or ynoates to siloxy(trialkoxy)ethene (KSA) is described. A siloxy group on the KSA directs the perfect regioselectivity, allowing rapid construction of various functionalized cyclobutenedione derivatives.

Spontaneous Cardiac Hypertrophy in a Crl:CD(SD) Rat.

Cardiac hypertrophy was observed in a 9-week-old Crl:CD(SD) rat that died unexpectedly. The animal was allocated to the control group of a toxicity study, and no abnormalities in its general conditions, body weight or food intake were observed. Necropsy revealed an increase in heart weight. Gross examination indicated cardiac enlargement with thickening of the right and left ventricular walls. Histopathological examination revealed hypertrophy of the cardiomyocytes in the right and left ventricular walls and the interventricular septum. Electron microscopic examination indicated bizarre nuclei and accumulation of an increased number of various sizes of mitochondria in the perinuclear region of the hypertrophied myocytes. Hypertrophied myocytes connected by intensely folded intercalated disks were also observed. Based on these findings, the animal was diagnosed with cardiac hypertrophy. This is the first case report of cardiac hypertrophy in this strain.

Microarray analysis of laser capture microdissected substantia nigra pars compacta after a single administration of MPTP in common marmosets.

Gene expression profiling was performed in the laser capture microdissected substantia nigra pars compacta (SNc) after a single administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in common marmosets. RNA from substantia nigra pars compacta tissue underwent a double linear amplification and was then applied to a whole human genome oligo microarray. Since the number of TH-immunoreactive cells in the SNc decreased to 50% of that in the control after 14 days and since our previous study showed that down-regulation of mRNA levels precedes cell death, genes for which mRNA levels were below 50% of levels in the vehicle-treated control throughout the experimental period were defined as being vulnerable to MPTP in the present study. There were 48 genes that satisfied this condition. In order to elucidate mechanisms of dopaminergic cell death in Parkinson's disease, extensive gene expression studies at various time points after MPTP and with various MPTP doses are required.

Classification of antipsychotic drugs by gene expression in the frontal cortex of mice.

Antipsychotic drugs are classified as typical and atypical based on extrapyramidal effects. However, since the frontal cortex is one of the most important regions for antipsychotic actions, this study attempted to classify antipsychotic drugs based on gene expression in the frontal cortex. Chlorpromazine and thioridazine were selected as typical antipsychotics, and olanzapine and quetiapine as atypical antipsychotics. Since these drugs have similar chemical structures, the effect of the basic structure on gene expression can be eliminated. Cluster analysis of microarray experiments separated 4-drug-administered mice into chlorpromazine-quetiapine and thioridazine-olanzapine groups. This classification scheme is different from that which is based on criteria currently used to group the typical and atypical drugs and suggests that antipsychotic drugs can be further separated into multiple groups.

Administration of haloperidol with biperiden reduces mRNAs related to the ubiquitin-proteasome system in mice.

In order to find molecules affected by administration of an antipsychotic drug with an antimuscarinic drug, which is a common prescription used to prevent extrapyramidal adverse effects caused by the antipsychotic drugs, gene expression profiling in the frontal cortex was studied in mice. After 14 days of administration with 2 mg/kg haloperidol, a typical antipsychotic drug, and 2 mg/kg biperiden, a high-affinity antagonist for muscarinic receptors in the brain, approximately 500 mRNAs related to synaptic function were investigated. The levels of the mRNAs related to the ubiquitin-related systems were significantly reduced after the combined administration. However, the separate administration of either haloperidol or biperiden had little effect on the levels of the mRNAs. This result suggests that coadministration of haloperidol and biperiden specifically affects the ubiquitin-related system.

Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5'-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.

Antinociceptive mechanism of L-DOPA.

The mechanism of L-DOPA for antinociception was investigated. Nociceptive behaviors in mice after an intrathecal (i.t.) administration of substance P were evaluated. L-DOPA (i.t.) dose-dependently attenuated the substance P-induced nociceptive behaviors. Co-administration of benserazide (i.t.), a DOPA decarboxylase inhibitor, abolished the antinociceptive effect of L-DOPA. The L-DOPA-induced antinociception was antagonized by sulpiride, a D2 blocker, but not by SCH 23390, a D1 blocker. These results suggest that L-DOPA relieves pain after conversion to dopamine, with the dopamine sedating pain transmission by way of the dopamine D2 receptor.

[The regulatory mechanism for neuron specific expression of PACAP gene].

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, is present abundantly in the central nervous system. In the 5'-flanking region of the PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element (NRSE). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. NRSE is a crucial negative-acting DNA regulatory element for neuron-specific gene expression. NRSE acts through the transcription factor known as neural-restrictive silencer factor (NRSF). In non-neuronal cells, NRSF suppresses the expression of neuron-specific genes. On the other hand, in neuronal cells, NRnV, a NRSF truncated form, repress their expressions in a dominant negative manner. The electrophoretic mobility shift assay with 3T3 cells extract demonstrated the identical complexes among NRSLE-1, NRSLE2, and the NRSE of rat type II sodium channel gene. In the luciferase reporter assay, NRSLEs suppressed SV40 promoter activity in 3T3 cells, but not in PC12 cells. RT-PCR analysis revealed that PACAP and NRnV mRNAs are expressed in neuronal cells (differentiated PC12), but not in non-neuronal cells (3T3 or C6). These results suggested that the NRSE-NRSF system might be involved in the regulatory mechanism of neuron-specific expression of the PACAP gene.

Gene expression profiling in the midbrain of striatal 6-hydroxydopamine-injected mice.

In order to clarify mechanisms underlying dopaminergic neuronal death in Parkinson's disease (PD), a gene expression profiling study was performed in a rodent model of PD. In this model, mice are intrastriatally injected with 6-hydroxydopamine (6-OHDA) and dopaminergic neurons in the substantia nigra (SN) gradually die by retrograde degeneration. The SN were removed 2 h, 24 h, or 14 days after 6-OHDA administration. Levels of mRNAs related to cell death or survival were quantified using adaptor-tagged competitive PCR (ATAC-PCR). The cyclin D1 gene showed an immediate increase in mRNA expression. After 24 h, when dopaminergic neurons were under intense degeneration, levels of caspase 8 mRNA and p53 apoptosis effecter related to pmp 22 (PERP) mRNA increased and, conversely, FAS mRNA decreased. After 14 days, when the degeneration was attenuated, levels of PERP mRNA and serum- and glucocorticoid-regulated kinase (SGK) mRNA still increased. SGK has a similarity to AKT, which is an important molecule involved in nerve growth factor signal transduction. AKT mRNA levels are low in dopaminergic neurons. These results suggest that an increase in cyclin D1 mRNA triggers dopaminergic neurons to enter an abnormal cell cycle, which leads to neuronal degeneration and cell death, possibly induced by PERP and caspase 8. In addition to cell death-related genes, several survival-related genes are activated. SGK might function as a key enzyme for the survival of dopaminergic neurons.