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PubMed1989

The research center for ultra-high voltage electron microscopy at Osaka University.

High-voltage electron microscopy has shown itself advantageous for the study of natural science, including biology, but especially for materials science. The most important advantage for materials science is for in situ experiments about the detailed processes of the phenomena that occur in bulk materials. The present paper is mainly concerned with several types of in situ experiments that have been carried out in the Research Center for Ultra-High Voltage Electron Microscopy, Osaka University. The following subjects have been studied: a) fundamental problems, such as the conditions necessary for in situ experiments, functional features of specimen treatment devices, and the effects of electron irradiation; b) the dislocation behavior of crystals under various conditions; c) high-temperature behavior of refractory materials, mainly ceramic composites; d) new applications of electron irradiation effects, such as amorphization of crystalline materials and electron-irradiation-induced foreign-atom implantation; e) environment-matter interaction, mainly chemical amorphization of alloys; and f) future trends of the in situ experiment, such as combinations with Auger valency electron spectroscopy and high-resolution electron microscopy.

Crystallization↗
PubMed2005

A vertebrate homolog of the cell cycle regulator Dbf4 is an inhibitor of Wnt signaling required for heart development.

Early stages of vertebrate heart development have been linked to Wnt signaling. Here we show in both gain- and loss-of-function experiments that XDbf4, a known regulator of Cdc7 kinase, is an inhibitor of the canonical Wnt signaling pathway. Depletion of endogenous XDbf4 protein did not disturb gastrulation movements or early organizer genes but resulted in embryos with morphologically defective heart and eyes and suppressed cardiac markers. These markers were restored by overexpressed XDbf4, or an XDbf4 mutant that inhibits Wnt signaling but lacks the ability to regulate Cdc7. This indicates that the function of XDbf4 in heart development is independent of its role in the cell cycle. Moreover, our data suggest that XDbf4 acts through the physical and functional interaction with Frodo, a context-dependent regulator of Wnt signaling. These findings establish an unexpected function for a vertebrate Dbf4 homolog and demonstrate the requirement for Wnt inhibition in early cardiac specification.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The transcription factor six1 inhibits neuronal and promotes hair cell fate in the developing zebrafish (Danio rerio) inner ear.

The developmental processes leading to the differentiation of mechanosensory hair cells and statoacoustic ganglion neurons from the early otic epithelium remain unclear. Possible candidates include members of the Pax-Six-Eya-Dach (paired box-sine oculis homeobox-eyes absent-dachshund) gene regulatory network. We cloned zebrafish six1 and studied its function in inner ear development. Gain- and loss-of-function experiments show that six1 has opposing roles in hair cell and neuronal lineages. It promotes hair cell fate and, conversely, inhibits neuronal fate by differentially affecting cell proliferation and cell death in these lineages. By independently targeting hair cells with atoh1a (atonal homolog 1a) knockdown or neurons with neurog1 (neurogenin 1) knockdown, we showed that the remaining cell population, neurons or hair cells, respectively, is still affected by gain or loss of six1 function. six1 interacts with other members of the Pax-Six-Eya-Dach regulatory network, in particular dacha and dachb in the hair cell but not neuronal lineage. Unlike in mouse, six1 does not appear to be dependent on eya1, although it seems to be important for the regulation of eya1 and pax2b expression in the ventral otic epithelium. Furthermore, six1 expression appears to be regulated by pax2b and also by foxi1 (forkhead box I1) as expected for an early inducer of the otic placode. Our results are the first to demonstrate a dual role for a member of the Pax-Six-Eya-Dach regulatory network in inner ear development.

Animals↗
PubMed2026

LncRNA RP11-708J19.2 promotes colorectal cancer progression by binding to SIRT7 via regulating H3K18ac.

Colorectal cancer (CRC) is a prevalent malignancy with a complex genetic basis. Recent genome-wide association studies (GWAS) have identified a susceptibility locus at 3p21.31, however, the functional SNP(s) underlying the association between the 3p21.31 region and CRC remain to be elucidated. In this study, we identified rs2101247 as the potential functional SNP and further demonstrated that rs2101247 is significantly associated with the expression of the nearby long non-coding RNA (lncRNA) RP11-708J19.2 (ENSG00000271161.1). Functional experiments showed that RP11-708J19.2 is upregulated in CRC tumor tissues, and its knockdown reduces cell viability while promoting apoptosis in SW1116 and HCT116 cell lines. Mechanistically, RP11-708J19.2 interacts directly with the deacetylase SIRT7, modulating histone H3K18 acetylation (H3K18ac). Specifically, RP11-708J19.2 knockdown leads to a significant upregulation of H3K18ac levels, implicating a SIRT7-mediated epigenetic pathway in CRC progression. Our findings elucidate a novel functional SNP-lncRNA axis that contributes to CRC pathogenesis, providing potential biomarkers for early detection and therapeutic targets for intervention.

Humans↗
PubMed1979

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.

4-Nitrophenylphosphatase↗
PubMed1996

Calcium pools, calcium entry, and cell growth.

The Ca2+ pump and Ca2+ release functions of intracellular Ca2+ pools have been well characterized. However, the nature and identity of Ca2+ pools as well as the physiological implications of Ca2+ levels within them, have remained elusive. Ca2+ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca2+ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca2+ pools, the levels of Ca2+ within Ca2+ pools, and how these intraluminal Ca2+ levels may be physiologically related to ER function. Experiments utilizing in situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluorescent Ca2+ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools within cells, and large changes in the levels of Ca2+ within pools following Insp3-mediated Ca2+ release. Such changes in Ca2+ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca(2+)-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca2+ changes with respect to cell growth. Emptying of pools using Ca2+ pump blockers can result in cells entering a stable quiescent G(o)-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca2+ pools return, and cells re-enter the cell cycle. During the Ca2+ pool-depleted growth-arrested state, cells express a Ca2+ influx channel that is distinct from the store-operated Ca2+ influx channels activated after short-term depletion of Ca2+ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca2+ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.

Animals↗
PubMed2005

The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma.

PURPOSE: Chemokine receptors are implicated in metastasis of several malignant tumors. This study was done to evaluate the contribution of chemokine receptors CXCR4 and CCR7 to metastasis of human nasopharyngeal carcinoma. EXPERIMENTAL DESIGN: Reverse transcription-PCR, immunohistochemistry, and flow cytometry were used to evaluate mRNA and protein expression of CXCR4 and CCR7 in nasopharyngeal carcinoma tumor tissues and cell lines. Chemotaxis assays were used to evaluate the function of CXCR4 in nasopharyngeal carcinoma cells. Antisense CXCR4 was used to inhibit receptor expression and to block metastasis of human nasopharyngeal carcinoma cells in vivo in athymic mice. RESULTS: CXCR4 protein was detected in tumor cells in 31 of 40 primary human nasopharyngeal carcinoma and in 13 of 15 lymph node metastases. CXCR4 transcripts were detected in eight CXCR4 protein-positive primary nasopharyngeal carcinoma tissues and seven nasopharyngeal carcinoma cell lines tested. On the other hand, the transcripts for CCR7 were detected only in four primary nasopharyngeal carcinoma tissues and in none of the nasopharyngeal carcinoma cell lines. In functional experiments, metastatic nasopharyngeal carcinoma cell lines that expressed high levels of CXCR4 were found to migrate in response to the CXCR4 ligand SDF-1alpha. Transfection of antisense CXCR4 in metastatic nasopharyngeal carcinoma cells inhibited the expression of CXCR4 and SDF-1alpha-induced cell migration in vitro and reduced the capacity of the tumor cells to form metastasis in the lungs and lymph nodes when injected in athymic mice. CONCLUSION: The expression of functional CXCR4 but not CCR7 is correlated with the metastatic potential of human nasopharyngeal carcinoma cells. Therefore, CXCR4 may be considered as a potential target for the prevention of nasopharyngeal carcinoma metastasis.

Animals↗
PubMed1999

Potential functional significance of brain-type and muscle-type nitric oxide synthase I expressed in adventitia and media of rat aorta.

Skeletal muscle and myocardium express microNOS I, an elongated splice variant of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothelial-type NO synthase, respectively. This study was designed to elucidate whether vascular smooth muscle also contains a constitutively expressed NO synthase isoform. In the rat, microNOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse transcription-polymerase chain reaction with primers flanking this insert and with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and microNOS I. RNase protection analyses with an antisense RNA probe overlapping the microNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of microNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and microNOS I showed a major band of the 160-kd NOS I and a lesser band of a slightly larger protein in endothelium-denuded aorta. Immunohistochemistry demonstrated low levels of NOS I/microNOS I immunoreactivity in the medial layer of rat aorta, whereas the endothelium expressed only NOS III immunoreactivity. When the adventitia also was removed, NOS I and microNOS I mRNA decreased markedly but remained detectable in the medial layer. In functional experiments with endothelium-denuded rat aortic rings (that contained no NOS III), contractions induced by KCl were markedly increased in the presence of the NOS inhibitor N(G)-nitro-L-arginine. These data demonstrate that 2 subforms of NOS I are expressed in nonendothelial components of rat aorta: NOS I and lesser amounts of microNOS I. Under certain conditions, this NOS I/microNOS I expression could serve as a backup system to the functionally predominant NOS III.

Animals↗
PubMed2002

Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats.

Expression of constitutive and inducible cyclooxygenase (COX-1 and COX-2, respectively) and the role of prostanoids were investigated in the aorta and mesenteric vascular bed (MVB) from the portal vein-ligated rat (PVL) as a model of portal hypertension. Functional experiments were carried out in MVB from PVL and sham-operated rats in the absence or presence of the nonselective COX inhibitor indomethacin or the selective inhibitors of COX-1 (SC-560) or COX-2 (NS-398). Western blots of COX-1 and COX-2 proteins were evaluated in aorta and MVB, and PGI(2) production by enzyme immunoassay of 6-keto-PGF(1alpha) was evaluated in the aorta. In the presence of functional endothelium, decreased contraction to norepinephrine (NE) and increased vasodilatation to ACh were observed in MVB from PVL. Exposure of MVB to indomethacin, SC-560, or NS-398 reversed the hyporeactivity to NE and the increased endothelial vasodilatation to ACh in PVL, with NS-398 being more potent than the other two inhibitors. Upregulation of COX-1 and COX-2 expressions was detected in aorta and MVB from PVL portal hypertensive rats, and increased production of 6-keto-PGF(1alpha) was observed in aorta from portal hypertensive rats. These results suggest that generation of endothelial vasodilator prostanoids, from COX-1 and COX-2 isoforms, accounts for the increased mesenteric blood flow in portal hypertension.

6-Ketoprostaglandin F1 alpha↗
PubMed2005

Alteration of reproductive hormone levels in pregnant sows induced by repeated ACTH application and its possible influence on pre- and post-natal hormone secretion of piglets.

Prenatal stress has been seen as a reason for reproductive failures in pig offspring mostly originated or mediated by changed maternal functions. Experiments were conducted in pregnant gilts (n=32) to characterize effects of elevated maternal glucocorticoids on the secretion of reproductive hormones (LH, progesterone) during the 1st (EXP 1), 2nd (EXP 2) and 3rd (EXP 3) trimester of pregnancy (TP). Transiently elevated cortisol release was repeatedly achieved by application of 100 IU adenocorticotropic hormone (ACTH) (Synacthen Depot) six times every second day beginning either on day 28 (EXP 1), day 49 (EXP 2) or day 75 of pregnancy (EXP 3). Glucocorticoid concentrations were examined in umbilical blood vessels of fetuses which mothers were subjected to ACTH at 2nd and 3rd TP (EXP 4). Furthermore, the pituitary function of newborn piglets of EXP 2 was checked by a LH-RH challenge test. In sows, LH concentrations were at low basal level (0.1-0.2 ng/ml) but with pulsatory release pattern during each TP. The number of LH pulses/6 h (LSM +/- SE) of saline treated Controls increased with ongoing pregnancy and decreased to the 3rd TP (1.3 +/- 0.2 in EXP 1 vs. 2.0 +/- 0.1 in EXP 2 vs. 1.4 +/- 0.1 in EXP 3, p<0.05). After ACTH treatment the number of LH pulses left unchanged in Experiments 1 and 2 (1.3 +/- 0.2 and 1.5 +/- 0.1) and decreased in EXP 3 (0.8 +/- 0.2, p<0.05). Differences (p<0.05) were obtained comparing the LH pulse number of ACTH and saline treated sows at the 2nd and 3rd TP. Moreover, areas under the curve (AUC) of each LH pulse and of LH over baseline were significantly reduced by treatment. Levels of progesterone increased (p<0.05) for 150 to 170 min after each ACTH application both in EXP 1 and EXP 2, but not in EXP 3. The mean progesterone concentration was different between trimesters, and ACTH and Controls (1st TP: 30.0 +/- 0.9 and 24.4 +/- 0.7 ng/ml; 2nd TP: 35.5 +/- 0.9 and 29.1 +/- 1.0 ng/ml; 3rd TP: 13.6 +/- 0.2 and 13.1 +/- 0.1 ng/ml; p<0.05). In fetuses (n=87) recovered 3 h after ACTH or saline (EXP 4), the plasma cortisol concentrations were significantly increased in umbilical vein (93.7 +/- 5.5 vs. 47.0 +/- 5.3 nmol/l) and artery (95.7 +/- 5.4 vs. 66.4 +/- 5.4 nmol/l), and in periphery (46.8 +/- 5.3 vs. 27.1 +/- 5.3 nmol/l) compared to controls. Plasma ACTH concentrations, however, did not differ in fetuses of both treatment groups. Postnatal LH-RH challenge tests (1st and 28th day post partum) induced LH surges in female piglets (n=67) both of ACTH and saline treated sows, but did not differ between groups (1st day: 7.2 +/- 0.8 vs. 8.1 +/- 0.7 ng/ml; 28th day: 10.5 +/- 1.7 vs. 13.6 +/- 2.2 ng/ml). However, basal LH of piglets whose mothers were submitted to ACTH during 2nd TP was lower on 1st day (1.7 +/- 0.2 vs. 2.3 +/- 0.2 ng/ml, p<0.05) but not on 28th day (1.0 +/- 0.2 vs. 1.1 +/- 0.2 ng/ml). However in both groups, the basal LH was always higher on 1st as on 28th day (p<0.05). Thus, chronic intermittent ACTH administration is able to influence the release pattern of maternal reproductive hormones. However, these findings demonstrate that these effects are dependent on the stage of pregnancy. Furthermore, it was shown that maternal cortisol can cross the placenta during gestation and thus may affect maternal-fetal interactions and, as a result, reproductive function of offspring.

Adrenocorticotropic Hormone↗
PubMed1999

Age-related changes of the nucleolar organizer regions without neuron loss in medial mamillary bodies (hypothalamus) in old rats.

Age-related morphological and functional changes were studied in the medial mamillary nuclei of the hypothalamus (MMN). The number of nerve cells and the volume of MMN were estimated in both groups of Wistar rats, adult and old (3 and 22 months, respectively) using stereologic methods such as the optical fractionator and Cavalieri's method respectively. The number of neurons and glial cells remain inalterable with ageing but there was an age-dependent reduction in MMN volume. In the second experiment, functional changes in the MMN neurons were observed although there was no loss in neuron number. Several functional parameters of the Ag-NORs were quantified by a computer analysis system: area and number of Ag-NORs per neuron; number of neurons with one or several Ag-NORs and also surface of neuronal nucleus. The present study shows how ageing causes volumetric and functional changes in the MMN whereas the number of neurons and glial cells remain unchanged in the mamillary region. All these results confirm the effects of age on proteic synthesis activity in neurons of the MMN, showing a decreased neuronal activity in this region.

Aging↗
PubMed2006

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

Base Sequence↗
PubMed1977

An in vitro study of functional maturation of murine thymus cells.

Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-theta serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed.

Animals↗
PubMed2001

sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish.

In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, we have isolated a zebrafish sprouty4 gene, based on its extensive similarities with the expression patterns of both fgf8 and fgf3. Through gain- and loss-of-function experiments, we demonstrate that Fgf8 and Fgf3 act in vivo to induce the expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses, Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, an opposite phenotype to the dorsalisation induced by overexpression of Fgf8 or Fgf3. Conversely we have shown that inhibition of Spry4 function through injection of antisense morpholino oligonucleotide leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, we show that Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, our analysis reveals that signaling through FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the regulation of bone morphogenetic protein (BMP) expression.

Amino Acid Sequence↗
PubMed2005

Identification of functional apple scab resistance gene promoters.

Apple scab (Venturia inaequalis) is one of the most damaging diseases affecting commercial apple production. Some wild Malus species possess resistance against apple scab. One gene, HcrVf2, from a cluster of three genes derived from the wild apple Malus floribunda clone 821, has recently been shown to confer resistance to apple scab when transferred into a scab-susceptible apple variety. For this proof-of-function experiment, the use of the 35S promoter from Cauliflower mosaic virus was reliable and appropriate. However, in order to reduce the amount of non-plant DNA in genetically modified apple to a minimum, with the aim of increasing genetically modified organism acceptability, these genes would ideally be regulated by their own promoters. In this study, sequences from the promoter region of the three members of the HcrVf gene family were compared. Promoter constructs containing progressive 5' deletions were prepared and used for functional analyses. Qualitative assessment confirmed promoter activity in apple. Quantitative promoter comparison was carried out in tobacco (Nicotiana glutinosa) and led to the identification of several promoter regions with different strengths from a basal level to half the strength of the 35S promoter from Cauliflower mosaic virus.

Ascomycota↗
PubMed2005

Ca2+ dynamics in salivary acinar cells: distinct morphology of the acinar lumen underlies near-synchronous global Ca2+ responses.

In salivary acinar cells, the pattern of the Ca2+ signals that regulates fluid and enzyme secretion has yet to be resolved, as there are conflicting reports in the literature. We have used a two-photon technique to directly visualize the acinar cell lumen in living fragments of exocrine tissue and simultaneously recorded agonist-induced changes in intracellular Ca2+. We show near-synchronous global Ca2+ responses in submandibular acinar cells, distinct from the typical apical to basal Ca2+ wave usually seen in rodent pancreatic acinar cells. In an effort to explain the basis of these near-synchronous global Ca2+ responses we used immunocytochemical experiments to localize luminal proteins and inositol trisphosphate receptors (InsP3Rs) in tissue fragments. Zona occludens 1 (ZO-1), a tight junction protein, shows that individual submandibular acinar cells are often nearly completely encircled by a narrow luminal structure. By contrast, in pancreatic fragments, ZO-1 staining shows short luminal branches terminating abruptly at the apical pole of single acinar cells. Co-immunostaining of InsP3Rs type 2 and type 3 showed them in the same region as ZO-1 in both exocrine tissues. Functional experiments showed that the near-synchronous global Ca2+ responses were still observed in the absence of extracellular Ca2+ and also in the presence of ryanodine. We conclude that the elaborate luminal region of submandibular cells leads to a hitherto unrecognized extensive distribution of InsP3Rs in a band around the cell and that this underlies the near-synchronous global Ca2+ response to agonists. We suggest that this may be a structural adaptation in submandibular cells to support the copious amounts of fluid secreted.

Animals↗
PubMed1993

The pharmacology of RS-15385-197, a potent and selective alpha 2-adrenoceptor antagonist.

1. RS-15385-197 ((8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro- 3-methoxy-12-(methylsulphonyl)-6H-isoquino [2,1-g][1,6]-naphthyridine) was evaluated in a series of in vitro and in vivo tests as an antagonist at alpha 2-adrenoceptors. 2. RS-15385-197 had a pKi of 9.45 for alpha 2-adrenoceptors in the rat cortex (pA2 in the guinea-pig ileum of 9.72), whereas the 8aS, 12aR, 13aR enantiomer, RS-15385-198, had a pKi of only 6.32 (pA2 6.47) indicating a high degree of stereoselectivity. The racemate RS-15385-196 had a pKi of 9.18. 3. RS-15385-197 showed unprecedented alpha 2 vs. alpha 1 adrenoceptor selectivity in vitro. In the rat cortex, RS-15385-197 had a pKi of 9.45 in displacing [3H]-yohimbine and 5.29 in displacing [3H]-prazosin (alpha 2/alpha 1 selectivity ratio in binding experiments > 14000). The compound had a pA2 of 9.72 as a competitive antagonist of the inhibitory effects of UK-14,304 in transmurally-stimulated guinea-pig ileum and 10.0 against BHT-920-induced contractions in dog saphenous vein (DSV); this latter value was unaltered by phenoxybenzamine. An apparent pKB of 5.9 was obtained against cirazoline-induced contractions in DSV, whilst a pA2 of 6.05 was obtained against phenylephrine-induced contractions in the rabbit aorta (alpha 2/alpha 1 selectivity ratio in functional experiments > 4000). 4. RS-15385-197 was highly selective for alpha 2-adrenoceptors over other receptors: the compound showed low affinity for 5-HT1A (pKi 6.50) and 5-HT1D (pKi 7.00) receptor subtypes, and even lower affinity (pKi < or = 5) for other 5-HT receptor subtypes, dopamine receptors, muscarinic cholinoceptors, beta-adrenoceptors and dihydropyridine binding sites. RS-15385-197 was devoid of affinity for the non-adrenoceptor imidazoline binding site, labelled by [3H]-idazoxan, which provides further evidence that these sites are not related to alpha 2-adrenoceptors. In the DSV, contractile responses to 5-hydroxytryptamine (5-HT) were unaffected by a concentration of 1 microM RS-15385-197. 5. RS-15385-197 was non-selective for the alpha 2A- and alpha 2B-adrenoceptor subtypes in that the pKi for the alpha 2A-adrenoceptor in human platelets was 9.90 and the pKi for the alpha 2B-adrenoceptor in rat neonate lung was 9.70. However, RS-15385-197 showed lower affinity for the alpha 2-adrenoceptor subtype in hamster adipocytes (pKi 8.38). 6. In anaesthetized rats, RS-15385-197 was a potent antagonist of the mydriasis response induced by UK-14,304 or clonidine (AD50 5 and 7 microg kg-1, i.v., respectively; 96 microg kg-1, p.o.) and of UK-14,304-induced pressor responses in pithed rats (AD50 7 microg kg-1, i.v.); the compound therefore is both centrally and orally active. Even at a high dose (10 mg kg-1, i.v.), RS-15385-197 did not antagonize pressor responses to cirazoline in pithed rats, indicating that the selectivity for alpha2 vs. alpha1-adrenoceptors was maintained in vivo.8 RS-15385-197 is therefore a very potent, selective, competitive alpha2-adrenoceptor antagonist, both in vitro and in vivo, is orally active and readily penetrates the brain. It will thus be a powerful pharmacological tool for exploring the various physiological roles of alpha2-adrenoceptors.

Administration, Oral↗
PubMed2002

TGTCACA motif is a novel cis-regulatory enhancer element involved in fruit-specific expression of the cucumisin gene.

Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a beta-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides -234 and -214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATATGATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions -254 and -215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.

Base Sequence↗