Sample collection from asteroid (162173) Ryugu by Hayabusa2: Implications for surface evolution.

The near-Earth asteroid (162173) Ryugu is thought to be a primitive carbonaceous object that contains hydrated minerals and organic molecules. We report sample collection from Ryugu's surface by the Hayabusa2 spacecraft on 21 February 2019. Touchdown images and global observations of surface colors are used to investigate the stratigraphy of the surface around the sample location and across Ryugu. Latitudinal color variations suggest the reddening of exposed surface material by solar heating and/or space weathering. Immediately after touchdown, Hayabusa2's thrusters disturbed dark, fine grains that originate from the redder materials. The stratigraphic relationship between identified craters and the redder material indicates that surface reddening occurred over a short period of time. We suggest that Ryugu previously experienced an orbital excursion near the Sun.

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.

Expression of dopamine transporter at the tips of growing neurites of PC12 cells.

The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.

Analysis of two pharmacologically predicted endothelin B receptor subtypes by using the endothelin B receptor gene knockout mouse.

1. This study was performed to clarify whether the endothelin (ET) receptor subtypes mediating two pharmacologically heterogeneous response to ETH receptor agonists in normal mice are the product(s) of a single ETB receptor gene. 2. Vasodilator responses to sarafotoxin S6c (S6c) in the thoracic aorta and contractile responses to ET-1 and IRL1620 in the stomach were examined in tissues from normal and ETB receptor gene knockout mice, in the absence and presence of an ETA receptor antagonist, BQ-123, or an ETA/ETB receptor antagonist, PD142893. 3. In the normal mouse aorta precontracted with phenylephrine, S6c (0.1-100 nM) caused concentration-dependent relaxations (pD2 = 8.4). BQ-123 had no effect on these responses. However, PD142893 almost abolished the relaxations induced by 0.1-300 nM S6c. 4. In aortae taken from ETB receptor gene knockout mice, S6c up to 1 microM failed to cause relaxations, confirming that ETB receptors are involved in mediating this response. 5. In normal mouse gastric fundus, 0.1 nM-1 microM ET-1, S6c or IRL1620 caused dose-dependent, BQ-123-insensitive contractions, which were much more resistant to PD142893 than S6c-induced relaxations of the aorta. The pD2 values for S6c in the absence and presence of PD142893 (10 microM) were 8.12 +/- 0.11 and 7.70 +/- 0.11, respectively. 6. In the gastric fundus of the ETB receptor gene knockout mouse, S6c and IRL1620 caused no contractions. ET-1 (0.1 nM-1 microM) caused contractions sensitive to both BQ-123 and PD142893, indicating that only ETA receptors mediate ET-1-induced contractions of the knockout mouse gastric fundus. 7. Since both the PD142893-sensitive vasodilator response of the aorta and the PD142893-resistant contractile response of the gastric fundus to S6c were completely absent in the ETB receptor gene knockout mouse, we conclude that the two pharmacologically heterogeneous responses to S6c are mediated by receptors derived from the same ETB receptor gene.

Localization of a 82 kDa protein in postsynaptic density and its association with cytoskeletons.

A fraction of synaptic junctional complex (SJC) was prepared from rat synaptosomes and served as antigen material to produce monoclonal antibodies (Mab) for examining the component proteins of the SJC. An antibody, Mab SJ-8, was obtained, which recognized a protein with a molecular weight of 82,000 Da in the SJC preparation by immunoblot analysis. The immunohistochemical localization of the 82 kDa protein was studied with the rat cerebellum. Mab SJ-8 labeled the peripheral areas of the Purkinje and granule cells. Small punctate areas were also stained in the molecular layer with SJ-8. Intracellular localization of the protein was examined with rat brain synaptosomes. Immunoelectron microscopy demonstrated that Mab SJ-8 strongly labeled the postsynaptic density (PSD) and also a fibrous network spreading out of it. However, the antibody did not label the pre- or post-synaptic membrane or the cleft material.

Characterization of [3H]5-hydroxytryptamine and [3H]spiperone binding sites in clathrin-coated vesicles from bovine brain.

Coated vesicles prepared from bovine brain cerebral cortex exhibited [3H]5-hydroxytryptamine (5-HT, serotonin) and [3H]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30-45 min at 30 degreesC, and was reversed by the addition of 100 microM 5-HT for [3H]5-HT binding or 10 microM ketanserin for [3H]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [3H]5-HT and [3H]spiperone with apparent Kd values of 2.4 and 1.75 nM, respectively. The binding of [3H]5-HT was displaced by 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [3H]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain alpha-subunits of GTP-binding proteins, Galphas, Galphai2, Galphai3, Galphao and Galphaq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.

Heterogeneity of endothelin ETA receptor-mediated contractions in the rabbit saphenous vein.

The involvement of endothelin ETA receptors in endothelin-1-induced contractions of the rabbit saphenous vein was studied. After desensitization of endothelin ETB receptors by pretreatment with sarafotoxin S6c, endothelin-1 and sarafotoxin S6b and a high concentration of endothelin-3 caused dose-dependent contractions. However, endothelin-1-induced contractions were much less sensitive to an endothelin ETA receptor antagonist, BQ-123 (cyclo (-D-Asp-L-Pro-D-Val-L-Leu-D-Trp-)), than sarafotoxin S6b-induced responses. The pA2 values of BQ-123 for endothelin-1- and sarafotoxin S6b-induced contractions were 5.69 and 7.65, respectively. These results suggest pharmacological heterogeneity of endothelin ETA receptors in the rabbit saphenous vein.

Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling.

We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.

Inhibitory effect of leptophos on carboxylesterase (isocarboxazid amidase) in rat liver.

The organophosphate insecticide, leptophos, inhibited rat liver isocarboxazid amidase (ISOCase) activity to 20% of control at 5.0 mg/kg l h after administration, but at this dose brain cholinesterase (ChE) activity was not affected. The activity of ISOCase decreased to 29 and 0% of control 24 h after treatment with leptophos at doses of 2.5 and 5.0 mg/kg, respectively. With repeated administration of leptophos at a dose of 1 mg/kg for 10 days, ISOCase activity decreased to 34% of control on day 1 and the inhibition increased to 85% on day 10 without inhibition of brain ChE activity. After cessation of three successive daily doses (1 mg/kg/day), the ISOCase activity was gradually restored near to control levels in 8 days. Pretreatment with carboxylesterase inhibitors, triorthocresylphosphate (TOCP) and bis-p-nitrophenylphosphate (BNPP), potentiated the inhibition of brain ChE by leptophos, suggesting that ISOCase might take a role in leptophos detoxification.

Reduction of ochratoxin A toxicity in mice treated with phenylalanine and phenobarbital.

LD50 of ochratoxin A (OCT A) was estimated to be 29.4 mg/kg in intraperitoneal (i.p.) and 46.0 mg/kg in per os (p.o.) administration in ddY strain male mice. Acute toxicity of OCT A was reduced by simultaneous administration of phenylalanine or by pretreatment with phenobarbital (PB) for 1 week and the LD50 increased to 1.5-2.0 times control. Chromatographic analyses of OCT A and the metabolite, OCT alpha, extracted from urine and bile after administration of OCT A, indicated that amounts of OCT A and OCT alpha decreased in the urine and increased in the bile of PB-pretreated mice, suggesting that a change in metabolism of OCT A could cause the decrease in the toxicity of OCT A in PB-pretreated mice.

The mode of action of ochratoxin A in acute enteritis in rats.

The mode of action of ochratoxin A(OCT A) was studied in male Wistar rats in connection with the development of acute enteritis. Acute enteritis in the duodenum and jejunum identical with that induced by oral administration was also induced by parenteral application at the dose level of 15 mg/kg OCT A and was completely inhibited by ligation of the choledochus. Direct application of OCT A into the jejunal blind sac lumen constructed by two ligations induced severe inflammation in situ and also revealed remote action to the duodenum and jejunum where separated from the blind sac by ligation. This remote action was inhibited by ligation of the choledochus. These results clearly demonstrated the enterohepatic circulation of OCT A. The ileal injection also revealed remote action of OCT A, although no pathologic change was caused in the ileal mucosa. The results obtained suggest that the enteritis may be induced by direct exposure of OCT A to the intestinal mucosa without metabolic activation, although certain participation of ochratoxin alpha to accelerate the inflammation was suspected.

[Anesthesia for a patient with heparin-induced thrombocytopenia: a case report].

A 68 year-old man underwent a femoral-popliteal bypass surgery. He was diagnosed as heparin-induced thrombocytopenia (HIT) by platelet aggregation test when he underwent CABG 4 years ago. For the present surgery we administered nafamostat mesilate and urokinase for anticoagulation during surgery instead of heparin. After the operation, laboratory studies showed no thrombocytopenia.

[The effects of preoperative drinking and H2 blocker on gastric acid secretion].

We studied the effects of preoperative drinking and H2 blocker on gastric acid secretion in 63 patients (ASA I-II, > 18yrs) scheduled for afternoon surgery. Group A (n = 20), as a control, was not permitted to eat and drink from 9 pm, the day before surgery, and was then given 500 ml of maintainance fluid before anesthesia. Group B (n = 20) fasted from 9 pm the day before surgery, and was allowed to drink clear fluids until 2hs before anesthesia. Group C (n - 23) followed the same guidelines as group B, and was given famotidine (20mg) orally at 9 pm the day before and 2hs before anesthesia. After induction, a Salem sump tube was inserted into the stomach and a gastric fluid aspiration was performed. The fluid volume and pH were measured after collection. Gastric pH was significantly higher (P < 0.001) in group C (6.4 +/- 0.9) than in groups A (3.1 +/- 1.8) and B (2.7 +/- 1.8). Fluid volume was similar in each group (A; 11 +/- 9/B; 12 +/- 9/C; 12 +/- 13ml). The dilution of gastric acid by the ingested fluid was not observed. We conclude that preoperative drinking does not affect gastric contents in elective operative patients. To reduce the risk of developing aspiration pneumonia, we recommend that every patient should receive an oral H2 blocker.

Expression of a dopamine transporter in PC12h cells: an immunohistochemical study with an antipeptide antibody.

Two kinds of oligopeptides, based on the amino acid sequences of No. 217-232 and 374-387 of a rat dopamine transporter, were designed and chemically synthesized. Five clones of the monoclonal antibodies against these peptides were produced with the in vitro immunization method. Two of them have recognized a protein of M(r) approximately 85,000 in a synaptosomal fraction of the rat striatum. It is probable that the protein M(r) approximately 85,000 corresponds to a dopamine transporter in the nerve terminal of the dopamine neurons in the striatum. The expression of dopamine transporter was immunohistochemically examined with these antibodies in PC12h cells. The immunoreactivity was detected on the surface membrane of the cells.

[Loneliness and self-consciousness in high-school students].

To examine relationships between loneliness and various aspects of self-consciousness in high-school students, UCLA Loneliness Scale (Russell, Peplau, & Cutrona, 1980), Self-Esteem Scale (Rosenberg, 1979), Self-Consciousness Scale (Fenigstein, Scheier, & Buss, 1975), Self-Monitoring Scale (Synder, 1974), and a High-School Life Questionnaire were administered to the first grade students in a high school (N = 182). Loneliness (alpha = .885), higher for males than for females, was significantly correlated with various aspects of their high-school lives. Loneliness was negatively correlated with self-esteem and self-monitoring, and was positively correlated with social anxiety. Only for males, a positive correlation was obtained between loneliness and private self-consciousness. Discriminant analysis and other correlational analyses also suggested that loneliness in males was related to various aspects of self-consciousness.

Estimation of harmine and its derivatives by HPLC: correlation of brain harmine levels with jumping behavior in rats.

Brain harmine was simply and sensitively determined by high performance liquid chromatography (HPLC), and the relationship between brain harmine concentrations and the jumping behavior induced by harmine and apomorphine was investigated. The concentration of harmine and the peak height in HPLC showed a good correlation, and the detection limit was 0.05 pmole harmine. In rats treated with 2, 5 and 10 mg/kg of harmine, the concentrations of harmine in the brain cortex were 9.0, 21.3 and 43.1 nmole/g tissue (wet wt.), respectively. The regional brain harmine concentrations and the jumping activity measuring by a scoring system increased with increasing doses of harmine administered, and there was a direct relation between the harmine concentrations and the jumping activity. This relation was observed in washed membranes in which about 40% of the harmine in whole homogenate was present. Tissue subfractionation showed that 30% of the harmine in whole brain homogenates was in the P2 fraction, and of this, 70% was located in the synaptosomes. The brain homogenate did not metabolize harmine and no metabolite was detected in the brain 10 min after treatment with harmine and apomorphine. These results suggest that harmine itself, but not its metabolites, may be responsible for inducing the jumping behavior and that the jumping activity is correlated with brain harmine concentrations.