Differential kinetics of oxytocin and vasopressin heteronuclear RNA expression in the rat supraoptic nucleus in response to chronic salt loading in vivo.

Previous studies have shown that the secretion of oxytocin and vasopressin from the posterior pituitary always accompanies systemic hyperosmotic stimuli in rats, and that oxytocin and vasopressin mRNAs consistently increase in response to prolonged hyperosmotic stimuli. Hence, it has been widely interpreted that oxytocin and vasopressin secretion and gene expression are closely coupled. In the present study, we used both vasopressin and oxytocin intron- specific probes to measure vasopressin and oxytocin heteronuclear RNA (hnRNA) levels, respectively, by in situ hybridisation in the rat supraoptic nucleus (SON) in conjunction with radioimmunoassays of vasopressin and oxytocin peptide levels in plasma and in the posterior pituitary in normally hydrated rats and after 1-5 days of salt loading. Increased oxytocin secretion in response to hyperosmotic stimuli exceeded vasopressin secretion at every time point studied. Vasopressin hnRNA in the SON increased to near maximal levels within minutes after the hyperosmotic stimulus, and was maintained throughout all 5 days of salt loading. By contrast, oxytocin hnRNA did not significantly change from control levels until approximately 2 days after hyperosmotic stimulation, and was not maximal until 3 days. In summary, increases in oxytocin gene transcription in response to osmotic stimuli are dramatically delayed compared to increases in vasopressin gene transcription under the same conditions. These data indicate that oxytocin gene transcription is not as closely correlated with pituitary peptide secretion as is vasopressin gene transcription, and suggests that there is a fundamental difference in excitation-secretion-transcription coupling mechanisms that regulate these two closely related genes in the rat magnocellular neurones in the SON.

The effects of GH-releasing hormone/somatostatin on the 5'-promoter activity of the GH gene in vitro.

The two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF), are known to regulate GH secretion. However, the effects of these hormones on GH gene expression are not completely clear, partly because of the lack of appropriate host cells maintaining the original characteristics of the somatotroph. Since MtT/S, a pure somatotroph cell line, has become available, the effects of GHRH and SRIF on GH gene transcription have been studied using a subclone of MtT/S (MtT/SGL), in which the GH gene 5'-promoter-luciferase fusion gene was stably incorporated. The expression of GHRH receptor and SRIF receptor subtypes was also studied by RT-PCR. The results showed that MtT/SGL cells intrinsically expressed the functional GHRH receptor and all of the SRIF receptor subtypes. The expression of GHRH receptor was markedly enhanced by glucocorticoid pretreatment and, in the presence of corticosterone and 3-isobutyl-1-methylxanthine, GHRH (at or above 100 pM) stimulated GH gene 5'-promoter activity in a dose-dependent manner. On the other hand, SRIF (100 nM) significantly antagonized the effect of GHRH, which was completely reversed by pretreatment with pertussis toxin (50 ng/ml). Taken together, the present data indicated that both GHRH and SRIF are involved in the transcriptional regulation of the GH gene, and that the effect of SRIF is mediated through pertussis toxin-sensitive G protein. The MtT/SGL cell line is a good in vitro model for studying the molecular mechanisms of GH gene transcription by GHRH and/or SRIF.

Homeobox protein Gsh-1-dependent regulation of the rat GHRH gene promoter.

Although GHRH is known to play a pivotal role in the regulation of the GHRH-GH-IGF-I axis, the molecular mechanism of GHRH gene expression has not yet been examined. Here we studied the transcriptional regulation of the GHRH gene 5'promoter using an in vitro experimental model system. We especially focused on the role of homeobox transcriptional factor Gsh-1, because a dwarf phenotype and abolished GHRH expression was observed in Gsh-1 knockout mice. First, we cloned human Gsh-1, which showed 87.3% homology with mouse Gsh-1 at the nucleotide level. When the 5'-promoter region of the rat GHRH gene was introduced into the human placental cell line JEG-3, in which we found the endogenous expression of Gsh-1 as well as GHRH mRNA, substantial transcriptional activity of the promoter was recognized. Promoter activity was further enhanced by overexpression of Gsh-1 protein, whereas it was substantially reduced by elimination of Gsh-1 binding sites. EMSA confirmed the actual binding of Gsh-1 on the multiple binding sites of GHRH gene promoter. Finally, coexpression of CREB-binding protein significantly enhanced the Gsh-1-induced GHRH gene expression, suggesting the cooperative role of the coactivator protein. Because Gsh-1 is found to be expressed in the hypothalamus of the adult rat, our data provide evidence that the Gsh-1 homeobox protein plays a key role in the expression of the GHRH gene.

Polyamine regulation of the rat pro-opiomelanocortin gene expression in AtT-20 cells.

Polyamines are a ubiquitous group of amines that play diverse biological roles. In the anterior pituitary, intracellular polyamine levels are reported to show diurnal changes, although the biological significance remains to be elucidated. In this study, we examined the effects of polyamines on the transcriptional activity of the rat pro-opiomelanocortin (POMC) gene using AtT20PL, a clone of the AtT20 cell line in which an approximately 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene was stably incorporated. The results showed that three representative polyamines (putrescine, spermidine and spermine) all stimulated POMC promoter activity in a time- and dose-related manner, spermine showing the most potent effect (maximum approximate three-fold increase). This effect was not observed under treatment with actinomycin D, suggesting the effect of polyamine at the transcriptional level. On the other hand, methylglyoxal bis (guanylhydrazone), an inhibitor of polyamine synthesis, showed the opposite effect, further supporting the positive role of intracellular polyamines. Taken together, our findings suggest that polyamines are involved in the regulation of POMC gene expression (especially in terms of diurnal changes) in corticotroph cells. The precise molecular mechanisms of polyamine effects await further research.

Effects of loperamide and other opioid-related substances on the transcriptional regulation of the rat pro-opiomelanocortin gene in AtT20 cells.

Although opioid peptides are involved in the regulation of the hypothalamic-pituitary-adrenal axis, their role in pro-opiomelanocortin (POMC) gene expression at the pituitary level is not known. We therefore examined the effects of opioid receptor agonists, including recently discovered endogenous opioid peptides, on POMC gene expression using the AtT20PL cell line, a subclone of AtT20 in which the rat POMC 5'-promoter-luciferase fusion gene was stably incorporated. The endogenous mu-opioid receptor agonists endomorphin 1 and 2 had no effect on either basal or corticotropin-stimulating-hormone-induced POMC expression. This was also the case with the delta-agonist BUBUC, the kappa-agonist U50488H and the orphan receptor agonist orphanin FQ. In contrast, the synthetic mu-agonist loperamide significantly inhibited basal and yet enhanced cAMP-induced POMC expression. The inhibitory effect of loperamide was mimicked by the calmodulin antagonist W7 and antagonized by the calcium channel blocker nifedipine, whereas neither the inhibitory nor the enhancing effect of loperamide was influenced by the opioid antagonist naloxone. These results suggest that the synthetic mu-agonist loperamide has a modulatory effect on the 5'-promoter activity of the POMC gene. This effect does not seem to be mediated through the classical mu-opioid receptor but rather in part through a calcium/calmodulin-related mechanism.

Involvement of upstream open reading frames in regulation of rat V(1b) vasopressin receptor expression.

The V(1b) vasopressin receptor, expressed mainly in the corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to approximately 5 kb from the translation start site), and characterized the 5'-flanking region of the rat V(1b) receptor gene. We identified the transcription start site by amplification of cDNA ends and found a new intron within the 5'-untranslated region (5'-UTR) by comparing the sequence with that of cDNA. We then confirmed that the obtained promoter indeed has transcriptional activity by use of the luciferase reporter in AtT-20 mouse corticotroph cells. Interestingly, there were five short upstream open reading frames (uORFs) located within the 5'-UTR that were found to suppress V(1b) expression. Subsequent mutational analyses showed that the two downstream uORFs have an inhibitory effect on expression in both homologous and heterologous contexts. Furthermore, the inhibition did not accompany a parallel decrease in mRNA, suggesting that the suppressive effect occurs at a level downstream of transcription. Taken together, our data strongly suggest that the expression of the V(1b) receptor is regulated at the posttranscriptional as well as transcriptional level through uORFs within the 5'-UTR region of the mRNA. Whether the uORF-mediated regulation of V(1b) expression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.

Antidiabetic sulfonylurea enhances secretagogue-induced adrenocorticotropin secretion and proopiomelanocortin gene expression in vitro.

The presence of high-affinity binding sites for antidiabetic sulfonylureas (SUs) and the expression of SU receptor (SUR) messenger RNA in the adenohypophyseal cells have recently been reported. In this study, we examined the effects of SU on POMC gene expression and ACTH secretion using the AtT20PL cell line, a subclone of AtT20 in which the rat POMC 5'-promoter-luciferase fusion gene was stably incorporated. A representative SU glibenclamide inhibited the basal POMC 5'-promoter activity. In contrast, glibenclamide enhanced forskolin- or CRH-induced POMC expression in a dose-dependent manner. Interestingly, the latter effect was not observed under treatment with 3-isobutyl-1-methylxanthine, a nonselective phosphodiesterase inhibitor. Furthermore, diazoxide, an opener of the ATP-sensitive K+ channel, only antagonized the suppressive effect of glibenclamide. Lastly, RT-PCR analysis showed that mouse SUR (but not SUR2) messenger RNA was expressed in this cell line. These results suggest that, in AtT20PL cells, SU has dual effects, i.e. a suppressive effect on basal POMC expression through diazoxide-sensitive (ATP-sensitive) K+-channel-mediated mechanism, and an enhancing effect on cAMP/protein kinase A-stimulated POMC expression through a different mechanism (probably mediated by phosphodiesterase). To our knowledge, this is the first report showing the effect of SU on the expression of the anterior pituitary hormone gene.

Molecular mechanisms of the negative effect of insulin-like growth factor-I on growth hormone gene expression in MtT/S somatotroph cells.

Although insulin-like growth factor-I (IGF-I) is shown to have a suppressive effect on GH gene expression at the pituitary level, its molecular mechanism has not yet been clarified. To study the issue, we established a new in vitro system using MtT/S, a recently established rat somatotroph tumor cell line that retains the basic characteristics of somatotroph function. Plasmids containing the GH 5' promoter (approximately 1.75 kb or shorter)-luciferase fusion gene were transfected stably or transiently into the cells, and the effect of IGF-I on the GH promoter activity was estimated by a luciferase assay. The results showed that IGF-I inhibited GH promotor activity (more than 50% suppression) in a time- and dose-related manner. IGF-I also inhibited GH secretion. A study using deletion mutants of the GH promoter revealed that the negative effect was maintained in the shortest construct (-80 to +6), suggesting that IGF-I-related factor is acting at the region very close to the minimal promoter. Interestingly, the negative effect was completely eliminated by a PI3 kinase inhibitor wortmannin (1 microM), whereas a MAP kinase inhibitor PD98059 (20 microM) or S6 kinase inhibitor rapamycin (10 nM) did not influence the effect. Our results suggest that IGF-I suppresses GH gene expression at the transcriptional level and that the PI3 kinase-mediated signaling pathway plays a major role in the negative effect of IGF-I. We believe that our system using MtT/S cells is an excellent experimental model system for studying the cellular and molecular mechanisms of the transcriptional regulation of GH in vitro.

Primary Ewing's sarcoma of the skull base with intracerebral extension--case report.

A 32-year-old female presented with an uncommon primary Ewing's sarcoma of the skull involving the middle cranial fossa with an extremely unusual extension into the cerebral parenchyma. She was treated with surgical excision, irradiation, and chemotherapy. Two years after surgery she demonstrated no evidence of local recurrence or distant metastasis. Ewing's sarcoma of the skull may achieve a better survival rate under adequate management than this lesion in other sites.

A newly designed nerve monitor for microneurosurgery: bipolar constant current nerve stimulator and movement detector with a pressure sensor.

The authors describe a newly designed nerve monitor which is useful for numerous microneurosurgical procedures. Standard bipolar forceps are used to apply constant current stimulation. Muscle contraction evoked by the stimulation is detected by a small disc-shaped pressure sensor taped to the overlying skin. The responses are monitored both quantitatively on a liquid crystal display and qualitatively through an on-off auditory signal. Surgery can proceed without interruption. This apparatus can safely and reliably monitor the facial nerve, nerves involved in eye movements, lower cranial nerves and spinal nerves. This portable system weights only 1.8 kg and can easily be used by a neurosurgeon.

Nucleotide sequence analysis of the simian virus 41 gene encoding the large (L) protein and construction of a phylogenetic tree for the L proteins of paramyxoviruses.

The complete nucleotide sequence of the simian virus 41 (SV41) large (L) protein gene was determined. The L gene spanned 6883 nucleotides including a putative trailer RNA, and the L mRNA contained a single large open reading frame encoding a polypeptide of 2269 amino acids. Dot-matrix comparisons under stringent conditions identified domains highly conserved among paramyxoviruses. Domain 3 is the most highly conserved, and has been hypothesized to be the RNA polymerase active site. A phylogenetic tree was constructed from the sequences of the L proteins of seven paramyxoviruses. SV41 was most closely related to human parainfluenza virus type 2 (HPIV-2), and SV41, HPIV-2 and SV5 form a subgroup. The intergenic sequences at the nucleocapsid protein-phosphoprotein and haemagglutinin-neuraminidase-L protein gene junctions, and the 5' trailer sequence of SV41 were also determined, and it was shown that the first 13 nucleotides of the 5' trailer sequence are complementary to those of the 3' leader sequence. The intergenic, and gene-start and -end sequences of SV41, HPIV-2 and SV5 are shown.

Molecular relationships between human parainfluenza virus type 2, and simian viruses 41 and 5: determination of nucleoprotein gene sequences of simian viruses 41 and 5.

The nucleotide sequences of cDNAs of the simian virus 5 (SV5) nucleoprotein (NP) gene, and the 3' end of the genome and NP gene of SV41 were determined. The open reading frames of the SV5 and SV41 NP genes encode polypeptides with Mrs of 56,582 and 60,575, respectively, values which are consistent with those estimated by SDS-PAGE. The NP of human parainfluenza virus type 2 (hPIV-2) was more closely related to that of SV41 (amino acid sequence identity 70.5%) than that of SV5 (57.0%); the amino acid sequence identity between the NPs of SV41 and SV5 was 63.3%. The sequence of the 3' end of the genome of SV41 showed a high level of similarity to that of hPIV-2, the terminal 18 nucleotides being identical. It is concluded from these findings that SV41 is related most closely to hPIV-2, even though SV5 had been thought to be an animal type of hPIV-2.

[An adult case of malignant choroid plexus papilloma in the lateral ventricle and the cerebellopontine angle revealed simultaneously].

An adult case of malignant choroid plexus papilloma is very rare. This report is an adult case of malignant choroid plexus papilloma revealed in the lateral ventricle and in the cerebellopontine (CP) angle simultaneously. A 37-year-old man was admitted to the hospital complaining of headache, nausea, and a floating sensation on August 29, 1984. Neurological examination on admission revealed bilateral papilledema, left dysmetria and horizontal nystagmus. CT scan revealed a slightly high density round mass in the right lateral ventricle and a cystic mass with mural nodule in the left CP angle. The intraventricular mass and mural nodule were enhanced moderately and homogeneously. The initial surgery was for removal of the CP angle tumor, and 8 days later removal of the lateral ventricle tumor was carried out. The histology of these tumors was the same and revealed malignant choroid plexus papilloma. Postoperative radiation therapy was carried out 70Gy to the brain (whole brain; 50Gy, focal; 20Gy) and 30Gy to the whole spine. About 2 years later paraparesis, lower cranial nerve palsy, and disturbance of consciousness had progressed gradually. He died of the severe recurrence of the tumor in the brain stem, and multiple dissemination in the spinal cord on September 6, 1987. There was no recurrence of tumor in the right lateral ventricle. This is a very rare case of malignant choroid plexus papilloma which was revealed in both the supra- and infratentorial regions simultaneously. They may have developed independently or they may have arisen through subarachnoid seeding. Radical removal of the tumor is important to prevent recurrence of malignant choroid plexus papilloma.

Localization of dural fistulas using metrizamide digital subtraction fluoroscopic cisternography.

Demonstration of the exact site of dural fistulas in cases of cerebrospinal fluid rhinorrhea is difficult. Previous reports have described the use of metrizamide cisternography combined with either hypocycloidal tomography or computerized tomography; however, direct, dynamic, real-time visualization of the fistula is difficult with instillation of a minimal dose of metrizamide using those methods. A digital video subtraction fluoroscopy system can visualize the actual site of the fistula directly and dynamically using only a small amount of metrizamide.

[The diagnostic method of localization of cerebrospinal fluid rhinorrhea by digital video subtraction system].

Demonstration of the exact site of the dural fistula in cerebrospinal fluid rhinorrhea is difficult. Previous reports present the methods of metrizamide cisternography combined with both hypocycloid tomography and computed tomography. But in these methods, direct, dynamic, actual and real-time visualization of the fistula with minimal dose of metrizamide is rather difficult. By using digital video subtraction system (Philips DVI-2CV), we could visualize the direct, dynamic and actual site of fistula with small amount of metrizamide instilled into the suboccipital subarachnoid space with the patient prone position. We report a successful case of traumatic cerebrospinal fluid rhinorrhea drained through the bony defect at the planum sphenoid into the posterior ethmoid sinus. This is the first report to deal with the usefulness of digital video subtraction system for exact localization of cerebrospinal fluid rhinorrhea. We emphasize the usefulness of metrizamide cisternography by the digital video subtraction system combined with the metrizamide computed tomographic cisternography for the precise localization of dural fistula.

[Extraneural metastasis of malignant glioma through a ventriculo-peritoneal shunt: growth in peritoneal cavity as ascitic form].

This is the first report of extraneural metastasis of malignant glioma through V-P shunt tube and growth in peritoneal cavity as ascitic form. The patient was a 43-year-old man who was admitted to our hospital with occipital headache. CT scan showed enhanced cystic tumor mass at left temporal lobe. Craniotomy and partial excision of the tumor was done and the histology of tumor tissue showed a malignant astrocytoma. Following this treatment, the patient received the adjuvant therapies of radiation, chemotherapy and immunotherapy with interferon, and also recraniotomy three times. In the mean time, a ventriculo-peritoneal shunt was set up for internal hydrocephalus. One month later, abdominal bulging appeared and yellowish ascites could be obtained with peritoneal tap. In the ascite, tumor cells with glial fibrillary acidic protein were observed at the concentration of 5-10 x 10(4) cells/ml. The patient died three months after extraneural metastasis to the abdominal cavity as ascitic form. At autopsy, solid metastatic mass lesion was not found in extraneural region include abdomen.

Regional distribution of dopamine and norepinephrine in canine cerebral arteries--effect of pre- or postganglionic sympathetic denervation.

The regional distribution of dopamine (DA) and norepinephrine (NE) was investigated in dog cerebral arteries using high performance liquid chromatography with electrochemical detection. The distribution patterns of these two amines differed and there was a wide fluctuation in the ratio between the amounts of DA and NE. The ratios of DA to NE in the anterior cerebral artery and the anterior inferior cerebellar artery were 2-4 times higher than in the basilar or middle cerebral arteries, thereby suggesting that DA plays a role other than that of precursor of NE. The concentrations of both amines following pre- or postganglionic sympathetic denervation (superior cervical ganglion) were investigated. After preganglionic denervation (decentralization), neither amine showed significant changes in concentration. Postganglionic denervation one week prior to sacrifice resulted in a reduction in the concentrations of both amines; however, decrease in the former was less. These results suggest that the origin of dopamine in the cerebral arteries differs from that of the sympathetic nerves, via the superior cervical ganglion.

Effects of ischemia on the removal of extracellular potassium in cat cortex during pentylenetetrazol seizures.

Changes in cortical extracellular potassium activity ([K+]0), NADH fluorescence, and oxygen consumption were studied in anesthetized cats during pentylenetetrazol seizures. The effects of partial ischemia induced by either hypotension or intermittent carotid artery occlusion on these parameters were investigated. Nonischemic seizures were characterized by gradual generalized decreases in cortical NADH fluorescence and increases in O2 consumption, along with rapid increases in [K+]0, which then usually fell slightly as the ictal discharge continued. Ischemic seizures, on the other hand, were accompanied by complex changes in NADH fluorescence, by smaller delayed maximal increases in O2 consumption that lasted beyond the end of ictal activity, and by more sustained increases in [K+]0. The decay of [K+]0 after the termination of seizures in both nonischemic and moderately ischemic animals was not a monoexponential function: plots of ln delta [K+]0 versus time showed an initial linear decline (of slope M1) that rather abruptly slowed (to slope M2) after 2 to 5 sec and then often increased to the original rate. Both M1 and M2 were proportionately decreased by ischemia. In addition, the rate of [K+]0 removal could be slowed by acute ischemia induced either during or after the end of ictal activity. The initial rate of postictal [K+]0 removal (M1) was found to be linearly and inversely related to the level of cortical NADH fluorescence at the time of seizure termination. The results of this study suggest that an O2-dependent transport mechanism plays a major role in the removal of [K+]0 during and following the termination of generalized pentylenetetrazol seizures in the cat.

Microneurosurgery for aneurysms of the basilar artery.

The authors report microsurgical treatment in 32 cases of basilar artery aneurysms, operated on with good results in 28 cases, fair results in one, and poor results in one; there were two deaths. Twenty-nine patients (91%) were able to return to social activities. Characteristics of the surgical techniques include 1) taking a transsylvian route; 2) retracting the M1 portion of the middle cerebral artery (occasionally the C1 portion of the internal carotid) medially with tapered brain retractors; and 3) approaching the aneurysm through and between perforators arising from the posterior cerebral artery in cases of high-placed basilar bifurcation. With regard to instrument improvements, tapered brain retractors, a multipurpose head frame, and bayonet clips (Sugita design) proved very helpful.