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Tumor-associated macrophages are associated with more malignant phenotypes of esophageal squamous cell carcinoma (ESCC) cells. Previously, an indirect co-culture assay of ESCC cells and macrophages was used to identify several factors associated with ESCC progression. Herein, a direct co-culture assay of ESCC cells and macrophages was established, which more closely simulated the actual cancer microenvironment. Direct co-cultured ESCC cells had significantly increased migration and invasion abilities, and phosphorylation levels of Akt and p38 mitogen-activated protein kinase (MAPK) compared with monocultured ESCC cells. According to a cDNA microarray analysis between monocultured and co-cultured ESCC cells, both the expression and release of S100 calcium binding protein A8 and A9 (S100A8 and S100A9), which commonly exist and function as a heterodimer (herein, S100A8/A9), were significantly enhanced in co-cultured ESCC cells. The addition of recombinant human S100A8/A9 protein induced migration and invasion of ESCC cells via Akt and p38 MAPK signaling. Both S100A8 and S100A9 silencing suppressed migration, invasion, and phosphorylation of Akt and p38 MAPK in co-cultured ESCC cells. Moreover, ESCC patients with high S100A8/A9 expression exhibited significantly shorter disease-free survival (P = 0.005) and cause-specific survival (P = 0.038). These results suggest that S100A8/A9 expression and release in ESCC cells are enhanced by direct co-culture with macrophages and that S100A8/A9 promotes ESCC progression via Akt and p38 MAPK signaling pathways., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: C3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear., Objective: We aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model., Methods: We analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer., Results: The CD8 + NKG2D + T and CD8 + T EM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4 + T EM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8 + T EM /T RM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c + cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8 + T cell percentages were upregulated in AA mice., Conclusion: These results suggest that the CD8 + and CD4 + T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model., Competing Interests: Declaration of Competing Interest All authors are employees of Maruho Co., Ltd., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Mucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood., Objective: The aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin., Methods: The protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER)., Results: In HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin., Conclusion: We found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling., Competing Interests: Declaration of Competing Interest The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. This study was funded by Maruho Co., Ltd., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor-associated macrophages (TAMs) promote tumor progression. The number of infiltrating TAMs is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC) patients; however, the mechanism underlying this phenomenon is unclear. cDNA microarray analysis indicates that the expression of chemokine (C-C motif) ligand 1 (CCL1) is up-regulated in peripheral blood monocyte-derived macrophages stimulated using conditioned media from ESCC cells (TAM-like macrophages). Here, we evaluated the role of CCL1 in ESCC progression. CCL1 was overexpressed in TAM-like macrophages, and CCR8, a CCL1 receptor, was expressed on ESCC cell surface. TAM-like macrophages significantly enhanced the motility of ESCC cells, and neutralizing antibodies against CCL1 or CCR8 suppressed this increased motility. Recombinant human CCL1 promoted ESCC cell motility via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway. Phosphatidylinositol 3-kinase or Akt inhibitors, CCR8 silencing, and neutralizing antibody against CCR8 could significantly suppress these effects. The overexpression of CCL1 in stromal cells or CCR8 in ESCC cells was significantly associated with poor overall survival (P = 0.002 or P = 0.009, respectively) and disease-free survival (P = 0.009 or P = 0.047, respectively) in patients with ESCC. These results indicate that the interaction between stromal CCL1 and CCR8 on cancer cells promotes ESCC progression via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway, thereby providing novel therapeutic targets., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

It is well known that the disruption of the mitochondrial respiratory components prolongs lifespan in many species. The mitochondrial stress response can lead to an increased survival rate through the restoration of the cellular homeostasis. Therefore, developing pharmacological interventions that induce mitochondrial stress response may be desirable to delay the onset of age-related diseases and promote a healthy life. In this study, we present chemical compounds, revealed by systematic screening of chemical libraries, which inhibit mitochondrial ATP synthesis in mammalian cells. Our study demonstrates that these compounds alter the body length and promote the oxidative stress response which leads to an increased longevity in Caenorhabditis elegans. Thus, our study identifies chemical compounds that may have potential therapeutic applications through affecting the mitochondrial function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although fibrosis is one of the most prominent pathologic features of preeclamptic (PE) placentas, its mechanism remains largely unknown. Consistent with previous reports, we observed overexpression of collagen; actin, α2, smooth muscle, aorta; connective tissue growth factor; and fibronectin in PE placentas compared with control ones. To investigate the mechanism of fibrosis in PE placentas, placental fibroblasts were isolated from PE placentas or normal pregnancies at delivery. The expression of fibrosis-related factors in fibroblasts was evaluated by real-time RT-PCR, Western blotting, enzyme-linked immunosorbent assay, and gene microarrays. An in vitro collagen gel contraction assay was also performed. Fibroblasts isolated from PE placentas showed higher expression levels of fibrosis-related factors compared with those from control ones. Global gene expression profiling of PE fibroblasts was contrasted with that of control ones and indicated an intimate association with transforming growth factor-β1 (TGFB1) signaling. Furthermore, the PE fibroblasts expressed abundant phosphorylated SMAD family member 2 and showed higher expression levels of target genes of TGFB1 signaling compared with the control ones. The PE fibroblasts also had a greater ability to contract compared with the control ones. Contractility also depended on TGFB1 signaling. Our results suggest that TGFB1 signaling is activated in the fibroblasts in PE placentas and that these active fibroblasts contribute to fibrosis., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Pathophysiological role for high-density lipoprotein (HDL) subclasses remains to be elucidated. Homogeneous assay for simultaneous measurements of apoE-deficient HDL-cholesterol (HDL-C), apoE-containing HDL-C, and total HDL-C is desired, because apoE plays important roles in lipid metabolism., Methods: The proposed assay consists of a primary reaction to remove non-HDL-C, a secondary reaction to measure apoE-deficient HDL-C, and a tertiary reaction to measure apoE-containing HDL-C. The assay is completed within 10 min. For control study, 13% polyethylene glycol precipitation assay and phosphotungstate-dextran sulfate-magnesium precipitation assay were carried out., Results: Good correlations between the control assays and the proposed assay was obtained in serum samples from patients without liver disease (n=33): r=0.987, 0.957, and 0.991 for apoE-deficient, apoE-containing, and total HDL-C, respectively. ApoE-containing HDL-C by the proposed method in healthy individuals (n=12) and patients with hyper-HDL-cholesterolemia (n=5) were 0.11±0.03 and 0.26±0.05 mmol/l (4.1±1.3 and 10.1±2.0 mg/dl), respectively. ApoE-containing HDL-C increased rapidly at >2.59 mmol/l (100 mg/dl) of total HDL-C, suggesting a unique regulating mechanism of apoE-containing HDL-C., Conclusions: The established homogeneous assay might be useful for clinical and epidemiological studies on apoE-deficient and apoE-containing HDL subclasses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: The purpose of this study was to assess the impact of a cognitive behavioral therapy (CBT) anxiety intervention on social phobia, social skill development, and self-concept., Method: Fifteen children with epilepsy and a primary anxiety disorder participated in a CBT intervention for 12 weeks plus a 3-month follow-up visit. Children were assessed at baseline, week 7, week 12, and 3 months post treatment to measure changes in social phobia using the Screen for Child Anxiety Related Emotional Disorders (SCARED). Self-concept was also assessed by using the Piers-Harris Children's Self-Concept Scale II (Piers-Harris 2)., Results: There was a significant reduction in symptoms of social phobia and improved self-concept at the end of the 12-week intervention and at the 3 month follow-up. Repeated measures ANOVA's of child ratings revealed significant change over time on the SCARED-Social Phobia/Social Anxiety subscale score (p=0.024). In terms of self-concept, significant change over time was detected on the Piers-Harris 2-Total score (p=0.015) and several subscale scores of Piers-Harris 2, including: Physical Appearance and Attributes (p=0.016), Freedom from Anxiety (p=0.005), and Popularity (p=0.003)., Conclusion: This pilot investigation utilized an evidenced based CBT intervention to reduce symptoms of social phobia, which in turn provided a vehicle to address specific social skills improving self-concept in children with epilepsy., (Copyright © 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

All bacteria are continuously exposed to environmental and/or endogenously active oxygen and nitrogen compounds and radicals. To reduce the deleterious effects of these reactive species, most bacteria have evolved specific sensor proteins that regulate the expression of enzymes that detoxify these species and repair proteins. Some bacterial transcriptional regulators containing an iron-sulfur cluster are involved in coordinating these physiological responses. Mechanistic and structural information can show how these regulators function, in particular, how chemical interactions at the cluster drive subsequent regulatory responses. The [2Fe-2S] transcription factor SoxR (superoxide response) functions as a bacterial sensor of oxidative stress and nitric oxide (NO). This review focuses on the mechanisms by which SoxR proteins respond to oxidative stress., (Copyright © 2013. Published by Elsevier Inc.)

Recent advances in perforator flap surgical techniques have allowed for safe and reliable autologous tissue transfer with minimal donor-site morbidity. Between April 2012 and January 2013, we performed immediate breast reconstruction using free medial circumflex femoral artery perforator (MCFAP) flaps in 15 patients after breast-conserving surgery. The flaps were harvested from patients in the lithotomy position, while a second surgical team simultaneously conducted tumour resection. Of the 15 procedures performed, three flaps were dissected as true perforator flaps, while 12 flaps were dissected as muscle-sparing perforator flaps. The mean flap weight was 138.5 g (range, 77-230 g) and the mean pedicle length was 5.7 cm (range, 3.0-9.0 cm). Recipient vessels for anastomosis were serratus branches in 10 patients, internal mammary vessels in three patients and thoracodorsal vessels in two patients. The mean duration of surgery was 6.74 h (range, 5.65-9.45 h). There were no major complications requiring surgical intervention. Researchers observed partial flap necrosis, which manifested as small firm lesions in two patients, as well as local wound infection and dehiscence in one patient, which resolved spontaneously. There were no instances of donor-site seroma formation or lymphoedema in any of the patients. An objective assessment of postoperative photographs showed that cosmetic results were mostly satisfactory. Donor-site scars along the medial groin crease were inconspicuous and readily concealed by clothing. Given its reliable vascularity and minimal donor-site morbidity, the free MCFAP flap can be a good alternative for partial breast reconstruction after breast-conserving surgery., (Copyright © 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Nuclear factor kappa B (NF-κB) is a transcription factor, which is translocated to the nucleus when activated. Herein, we demonstrate immunohistochemically that electrical, chemical, and thermal stimuli, applied to the skin of mice, all induced nuclear translocation and phosphorylation of NF-κB in dorsal root ganglia (DRG) neurons. The latency of this response was short, with effects observable in as little as 3min following stimulation. Few nuclear phospho-NF-κB-positive neurons were observed in DRG innervating unstimulated regions. These results suggest somatosensory stimuli quickly induce NF-κB-mediated gene transcription in DRG, and phospho-NF-κB could be a suitable histological marker for activated DRG neurons., (Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Background: Although β-blockers are used to prevent myocardial ischemia/reperfusion injury, the risk of heart failure has limited β-blocker therapy in patients with acute myocardial infarction. This study evaluated efficacy of intracoronary administration of the short-acting β-blocker, landiolol, during reperfusion in pigs with acute myocardial ischemia., Methods and Results: In the non-ischemic model landiolol administered into the left anterior descending coronary artery (LAD) inhibited in a dose-dependent fashion segmental wall thickening (SWT) in the anterior LV wall without altering SWT in the posterior LV wall and without prolonged depression of global LV function except for the highest dose. In the ischemic model with 60 min LAD occlusion followed by reperfusion the medium dose landiolol administered into the LAD 1 min before and for 10 min during reperfusion inhibited initial recovery of SWT in the anterior LV wall but improved SWT in this region and global LV function late after reperfusion. Ultrastructural studies showed inhibition of sub-sarcolemmal bleb formation by treatment with landiolol 10 min after reperfusion associated with the inhibition of CK-MB release and the reduction of infarct size. There was no significant difference in CK-MB release and infarct size between landiolol treatment for 10 min and 180 min during reperfusion., Conclusions: Selective and brief intracoronary administration of landiolol during reperfusion enhances myocardial salvage without causing deterioration of global LV function., (Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.)

A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of F₀F₁-ATP synthase (F₀F₁), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of β-subunit of F₀F₁, resulted in loss of ATP synthesis. Compared with the conventional procedures that need mitochondria fractionation and detergent, this assay is simple, sensitive and suitable for high-throughput analysis of genes and drugs that could affect mitochondrial functional integrity as represented by ATP synthesis activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Hyperventilation, with the resulting hypocapnia, reduces cerebral blood flow and causes slowing of the EEG activity. However, neuronal oscillating properties including the thalamocortical network during hyperventilation have not been elucidated. To assess these features provoked by hyperventilation, the present study examined quadratic phase coupling features by means of bicoherence analysis., Methods: Twenty-two patients were anaesthetized using sevoflurane 1.5% combined with remifentanil or epidural anaesthesia. After a stable normocapnic period, hypocapnia was induced by hyperventilation, and the raw EEG signals were collected. Bispectral analysis (bicoherence) and power spectrum analysis were performed before and after hypocapnia., Results: Mean (sd) peak bicoherence in the delta- area increased from 35.6 (10.9)% during normocapnia to 43.8 (10.9)% during hypocapnia (P<0.05), whereas mean (sd) peak bicoherence in the alpha area decreased from 42.8 (14.4)% during normocapnia to 37.5 (12.3)% during hypocapnia (P<0.05). Normalized power in the delta- frequencies on the power spectrum increased from 60.2 (13.1)% to 72.5 (12.7)% (P<0.05). Bispectral index and spectral edge frequency changed from 45.9 (7.0) to 40.1 (5.6) (P<0.05) and from 15.0 (2.3) to 14.0 (2.5) Hz (P<0.05), respectively. No significant differences in these values were observed between the two types of anaesthesia., Conclusions: Hypocapnia enlarged bicoherence growth in the delta- frequency range, suggesting the contribution of subcortical oscillating mechanisms in regulating EEG during hyperventilation.

The influence of the amounts of additives including 1-O-ethyl-3-n-butylcyclohexanol (OEBC), diisopropyl adipate (DIA), and isopropanol (IPA) on the penetration rate (R(p)) of ketoprofen from hydrogels through rat skin in vivo was investigated. Skin irritation evoked by the application of hydrogels was evaluated based on a microscopic observation of skin cross-sections. Both optimization techniques incorporating an artificial neural network (ANN) and a second-order polynomial regression analysis were applied to the optimization of ketoprofen hydrogel formulations. Findings indicated that the R(p) and total irritation score (TIS) of the skin were predicted quantitatively as a function of quantities of OEBC, DIA, and IPA, employing ANN. In contrast, the prediction ability of the polynomial regression equation was somewhat poorer compared with that of ANN. The observed results of R(p) and TIS in the optimal formulation coincided well with the predictions in the simultaneous optimization technique incorporating ANN., (Copyright 2001 Wiley-Liss, Inc.)

The present study was performed to examine the effects of regular (1 Hz) and modified rapid random stimulation (RRS) (6 and 12 Hz) on visual evoked potentials (VEPs), by simultaneously recording negative waves around 100 ms, wave IV-latency, positive waves around 60 ms, wave III-latency, and amplitudes calculated from peak to peak, without causing impairment of visual acuity, in 44 patients aged 5-17 years. The wave IV-latencies of VEPs obtained by 6 and 12 Hz RRS were easily determined, and the latencies were not significantly changed compared to those obtained by previous 1 Hz regular stimulation. On the other hand, the amplitudes decreased in a frequency-dependent manner (1 < 6 < 12 Hz). These results were found to be similar in both preschool and school children. The examination time of VEPs determined by RRS is one-tenth shorter than that of 1 Hz regular stimulation. Thus, this method has the benefit of shortening the examination time, which decreases fatigue and inattention of the subjects, suggesting that modified RRS is a practically useful method for children.

Objective: The prevalence of hepatitis G virus infection was evaluated in dental patients whose clinical laboratory test results were positive for hepatitis C virus antibody, hepatitis B virus surface antigen, or elevated serum alanine transaminase concentrations., Study Design: Frozen serum samples from patients with hepatitis C virus antibody (n = 63), hepatitis B virus surface antigen (n = 20), or alanine transaminase concentrations greater than 100 IU (n = 14) were assessed for GB virus C (GBV-C)/hepatitis G virus RNA by a reverse transcriptase-polymerase chain reaction., Results: Six of 63 patients with hepatitis C virus antibodies had serum hepatitis G virus RNA (9.5%), and 2 of 20 subjects with hepatitis B virus surface antigen had hepatitis G virus RNA (10.0%). None of 14 patients whose alanine transaminase concentration was greater than 100 IU/L had hepatitis G virus RNA. Of 4 subjects with both hepatitis C virus antibody and hepatitis B virus surface antigen, 2 had hepatitis G virus RNA (50%). In the total study population (N = 92), 6 subjects (6.5%) had hepatitis G virus RNA. All hepatitis G virus-infected patients also had hepatitis C virus antibody. Neither serum alanine transaminase nor aspartate transaminase concentrations were different between subjects with and subjects without hepatitis G virus RNA. The lack of a relationship between hepatitis G virus infection and elevation of alanine transaminase and aspartate transaminase might suggest that this virus is not truly a hepatitis virus., Conclusions: Hospitalized dental patients are infected with hepatitis G virus at a prevalence similar to or slightly higher than that seen in the general population. Dentists should pay close attention to infection control with respect to the potential new hepatitis virus known as hepatitis G virus.

The present experiments were performed to examine the effects of a new muscarinic M1-receptor agonist, (-)-YM796 ((-)-S-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro[4.5]decane L-tartrate monohydrate), on yawning and oxytocin secretion from the posterior pituitary gland in rats YM796, at doses of 2.5-50 mg/kg (SC), elicited yawning. The yawning response was markedly increased by pretreatment with a beta-adrenoceptor antagonist, pindolol (20 mg/kg, IP), which per se did not elicit yawning. The yawning induced by YM796 (10 mg/kg, SC) in combination with pindolol (20 mg/kg, IP) was inhibited by scopolamine (0.5 mg/kg, SC), a muscarinic receptor antagonist, and pirenzcpine (300 micrograms/ rat, ICV) and EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) (5 mg/kg, IP), muscarinic M1-receptor antagonists, but not by spiperone (0.5 mg/kg, SC), a dopamine D2-receptor antagonist, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) (100 micrograms/rat, ICV), a muscarinic M3-receptor antagonist, and [d(CH2)5, Tyr(Mc)2, Orn8]-vasotocin (100 ng/rat, ICV), an oxytocin receptor antagonist. YM796 at 2.5-50 mg/kg (SC) did not exert an action on prolactin levels but increased oxytocin secretion from the posterior pituitary gland in rats. This augmentation of oxytocin secretion by YM796 was inhibited by scopolamine (0.5 mg/kg, SC) and pirenzepine (3 mg/kg, SC), but not by mecamylamine (1 mg/kg, IP), a nicotinic receptor antagonist. The present findings obtained with YM796 suggest that the muscarinic M2-receptor stimulation participates in causing yawning behavior and oxytocin secretion in rats.

The present experiments were performed to examine the behavioral effects of OPC-14597, which acts on dopamine receptors in rats. OPC-14597 administered subcutaneously (SC) at doses of 0.1-5 mg/kg elicited yawning, as did OPC-4392 (0.5-2 mg/kg, SC) and (-)-3-PPP (2.5-10 mg/kg, SC). These yawning responses were blocked by intraperitoneal (IP) pretreatment with haloperidol (0.5 mg/kg) but were increased by pindolol (20 mg/kg, IP) or reserpine (5 mg/kg, IP), which per se did not elicit yawning. The yawning induced by talipexole, a selective dopamine D2 receptor agonist, was inhibited by OPC-14597 (0.5-5 mg/kg, SC) and (-)-3-PPP (10 mg/kg, SC). Apomorphine (0.5 mg/kg, SC), a dopamine D1/D2 receptor agonist, elicited stereotypy such as sniffing and licking but OPC-14597 (5-20 mg/kg, SC) did not induce this behavior. The stereotypy induced by apomorphine was inhibited not only by haloperidol (0.5 mg/kg, IP) and (-)-3-PPP (10 mg/kg, SC) but also by OPC-14597 (5-20 mg/kg, SC), without being affected by OPC-4392 (2 mg/kg, SC). In 6-hydroxydopamine (6-OHDA)-treated rats, apomorphine (0.5 mg/kg, SC) elicited rotation behavior whereas OPC-14597, OPC-4392 and (-)-3-PPP did not produce this behavior. These findings suggest that OPC-14597 provokes yawning without causing stereotypy and rotation but markedly antagonizes the talipexole-induced yawning and apomorphine-induced stereotypy, and that OPC-14597 thus exerts partial agonistic effects on yawning behavior but antagonistic effects on stereotypy in rats.

The present study was undertaken to investigate whether beta-adrenoceptors are involved in regulation of yawning responses to oxytocin and alpha-melanocyte-stimulating hormone (alpha-MSH) in rats. Oxytocin administered intracerebroventricularly (ICV) at doses of 50 and 100 ng/rat elicited yawning. alpha-MSH (20 micrograms/rat, ICV) elicited not only yawning but also stretching and body shaking. RS-86 (2-ethyl-8-methyl-2,8-diazaspiro-(4,5)-decan-1,3-dion hydrobromide), a putative muscarinic M1 receptor agonist, administered ICV at a lower dose of 100 micrograms/rat and subcutaneously (SC) at doses of 0.25-2.5 mg/kg also elicited yawning. The yawning responses produced by these agents were markedly increased by intraperitoneal (IP) pretreatment with a beta-adrenoceptor antagonist, pindolol (20 mg/kg), which per se did not elicit yawning. The yawning induced by oxytocin (50 ng/rat, ICV) plus pindolol, but not that by alpha-MSH (20 micrograms/rat, ICV) or RS-86 (0.5 mg/kg, SC) plus pindolol, was inhibited by [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (100 ng/rat, ICV), an oxytocin receptor antagonist. The yawning induced by oxytocin, alpha-MSH, or RS-86 administered in combination with pindolol was inhibited by scopolamine (0.5 mg/kg, SC), a mucarinic receptor antagonist, without being affected by spiperone (0.5 mg/kg, SC), a dopamine D2 receptor antagonist. The results suggest that the yawning produced by the neuropeptides oxytocin and alpha-MSH is modulated by beta-adrenoceptor activity in an inhibitory manner as that produced by muscarinic M1 receptor agonists, and that it involves cholinergic, but not dopaminergic, activation.

Two novel theta class glutathione S-transferases (GSTs), designated Yrs-Yrs' and Yrs'-Yrs', were isolated from rat liver cytosol and purified to homogeneity. Polyclonal antibody raised against the previously reported theta class GST Yrs-Yrs (Hiratsuka, A. et al., J. Biol. Chem. 265, 11973 (1990)) cross-reacted with GSTs Yrs-Yrs' and Yrs'-Yrs'. These three theta class GSTs had different pI values and were separated by chromatofocusing. The enzyme subunit Yrs' was separated from Yrs by reverse partition HPLC, but identical to Yrs in the first 37 N-terminal amino acid sequence. Like GST Yrs-Yrs, both GSTs Yrs-Yrs' and Yrs'-Yrs' were not retained on an S-hexyl-GSH affinity column and had little activity toward 1-chloro-2,4-dinitrobenzene. However, they showed potent activities toward the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene. GSTs Yrs-Yrs and Yrs'-Yrs' showed much higher GSH peroxidase activities toward arachidonate hydroperoxide than did rat liver alpha class GST Ya-Ya.

The mechanisms of leakage of intracellular enzymes, and especially the cytosolic and mitochondrial isozymes of aspartate aminotransferase (AST), in ischemic rat liver were studied. On recirculation of ischemic liver, cytosolic AST (cAST) promptly appeared in the blood. Release of cytosolic enzymes, including cAST and lactic dehydrogenase, resulted from disruption of blebs that protruded from parenchymal cells into the sinusoidal space. When these blebs were formed in ischemic liver, mitochondria still remained in core regions of the injured cells and were not found in the blebs. Consistent with this fact, mitochondrial AST (mAST) did not leak into the circulation from ischemic liver until most of the cAST had leaked out. This delayed leakage of mitochondrial enzymes was also consistent with the fact that the mitochondrial membranes maintained a diffusion barrier against matrix enzymes even after anoxia for 2 h, when their oxidative phosphorylation capacity had been lost. These results indicate that mitochondrial enzymes are liberated into the blood only after appreciable disintegration of the cells, probably necrosis, and that the cumulative activity of mAST in the blood should reflect the extent of necrosis in ischemic organs better than that of cAST.