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Social behavior, defined as any mode of communication between conspecifics is regulated by a widespread network comprising multiple brain structures. The anterior cingulate cortex (ACC) serves as a hub region interconnected with several brain regions involved in social behavior. Because the ACC coordinates various behaviors, it is important to focus on a subpopulation of neurons that are potentially involved in social behavior to clarify the precise role of the ACC in social behavior. In this study, we aimed to analyze the roles of a social stimulus-responsive subpopulation of neurons in the ACC in social behavior in mice. We demonstrated that a subpopulation of neurons in the ACC was activated by social stimuli and that silencing the social stimulus-responsive subpopulation of neurons in the ACC significantly impaired social interaction without affecting locomotor activity or anxiety-like behavior. Our current findings highlight the importance of the social stimulus-responsive subpopulation of neurons in the ACC for social behavior and the association between ACC dysfunction and impaired social behavior, which sheds light on therapeutic interventions for psychiatric conditions., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The number of patients with heart failure and related deaths is rapidly increasing worldwide, making it a major problem. Cardiac hypertrophy is a crucial preliminary step in heart failure, but its treatment has not yet been fully successful. In this study, we established a system to evaluate cardiomyocyte hypertrophy using a deep learning-based high-throughput screening system and identified drugs that inhibit it. First, primary cultured cardiomyocytes from neonatal rats were stimulated by both angiotensin II and endothelin-1, and cellular images were captured using a phase-contrast microscope. Subsequently, we used a deep learning model for instance segmentation and established a system to automatically and unbiasedly evaluate the cardiomyocyte size and perimeter. Using this system, we screened 100 FDA-approved drugs library and identified 12 drugs that inhibited cardiomyocyte hypertrophy. We focused on ezetimibe, a cholesterol absorption inhibitor, that inhibited cardiomyocyte hypertrophy in a dose-dependent manner in vitro. Additionally, ezetimibe improved the cardiac dysfunction induced by pressure overload in mice. These results suggest that the deep learning-based system is useful for the evaluation of cardiomyocyte hypertrophy and drug screening, leading to the development of new treatments for heart failure., (Copyright © 2022. Published by Elsevier Inc.)

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved pleiotropic neuropeptide, implicated in emotional stress responses and anxiety-related disorders. Here, we examined whether our recently developed small-molecule non-peptide PACAP receptor antagonists could ameliorate anxiety-like behaviors induced by acute restraint stress in mice. The antagonists PA-9 and its derivative PA-915 improved anxiety-like behaviors in mice subjected to restraint stress. An anxiolytic effect was observed with single acute dose, suggesting their fast-acting properties. PA-915 demonstrated a statistically significant anxiolytic effect whereas fluoxetine did not. These results indicate the potential of PAC1 antagonists as a novel treatment for anxiety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study., (Copyright © 2022. Published by Elsevier Inc.)

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by altered social communication, restricted interests, and stereotypic behaviors. Although the molecular and cellular pathogeneses of ASD remain elusive, impaired neural stem cell differentiation and neuronal migration during cortical development are suggested to be critically involved in ASD. ANK2, which encodes for a cytoskeletal scaffolding protein involved in recruiting membrane proteins into specialized membrane domains, has been identified as a high-confidence ASD risk gene. However, the role of ANK2 in early neural development remains unclear. In this study, we analyzed the role of ANK2 in the cerebral cortex of developing mouse using in utero electroporation. We provide evidence suggesting that ANK2 regulates neural stem cell differentiation and neuronal migration in the embryonic cerebral cortex, where Ank2 is highly expressed. We also demonstrated that Ank2 knockdown alters the expression of genes involved in neural development. Taken together, these results support the view that ANK2 haploinsufficiency in patients may impair neural development, resulting in an increased risk of ASD. Our study findings provide new insights into the molecular and cellular pathogenesis of ASD, given that among high-confidence ASD genes, ANK2 is rare in that it encodes for a scaffolding protein for the membrane protein complex required for neuronal functions., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder, characterized by impaired social interaction, repetitive behavior and restricted interests. Although the molecular etiology of ASD remains largely unknown, recent studies have suggested that de novo mutations are significantly involved in the risk of ASD. We and others recently identified spontaneous de novo mutations in PKD2, a protein kinase D family member, in sporadic ASD cases. However, the biological significance of the de novo PKD2 mutations and the role of PKD2 in brain development remain unclear. Here, we performed functional analysis of PKD2 in cortical neuron development using in utero electroporation. PKD2 is highly expressed in cortical neural stem cells in the developing cortex and regulates cortical neuron development, including the neuronal differentiation of neural stem cells and migration of newborn neurons. Importantly, we determined that the ASD-associated de novo mutations impair the kinase activity of PKD2, suggesting that the de novo PKD2 mutations can be a risk factor for the disease by loss of function of PKD2. Our current findings provide novel insight into the molecular and cellular pathogenesis of ASD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart., (Copyright © 2017. Published by Elsevier Inc.)

Cisplatin, known as an anticancer drug, has been widely used; however, diverse disadvantageous side effects, including appetite loss, afflict patients. Nesfatin-1/NucB2, discovered as an anorexic neuropeptide, is broadly expressed in the central nervous system (CNS) and peripheral organ. In the present study, we examined the effects of intraperitoneally (i.p.) administered cisplatin on central nesfatin-1/NucB2. Saline, as control, or cisplatin (6 mg/kg dissolved in saline) was i.p. administered in adult male Wistar rats (180-220 g). Cumulative food intake was remarkably suppressed for at least 24 h and body weight was significantly smaller at 24 h after i.p. administration of cisplatin compared to control group. At 90 min after i.p. administration, they were perfused, followed by carrying out double-immunohistochemistry for Fos and nesfatin-1/NucB2. The percentage of nesfatin-1/NucB2 immunoreactive neurons expressing Fos was marked increased in the hypothalamus and brainstem after i.p. administration of cisplatin. Intracerebroventricularlly administered nesfatin-1/NucB2-antisense resulted in a significant attenuation of decreased food intake for 2 h after i.p. administration of cisplatin compared to nesfatin-1/NucB2-missense treated group. These results suggest that i.p. administration of cisplatin activated, at least in part, nesfatin-1/NucB2 neuron in the CNS and may exert anorexigenic effects in rats., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glutamatergic N-methyl-d-aspartate (NMDA) receptors play critical roles in several neurological and psychiatric diseases. Blockade by noncompetitive NMDA receptor antagonist leads to psychotomimetic effects; however, the brain regions responsible for the effects are not well understood. Here, we determined the specific brain regions responsive to MK-801, a noncompetitive NMDA receptor antagonist, by mapping Arc expression as an indicator of neuronal activity using Arc::dVenus reporter mice. MK-801 increased dVenus expression predominantly in the orbitofrontal cortex (OFC) and, as expected, induced a marked hyperlocomotion. Local OFC lesions selectively attenuated the early phase (0-30 min) of MK-801-induced hyperlocomotion. Further, clozapine, an atypical antipsychotic, effectively attenuated both the MK-801-induced dVenus expression in the OFC and hyperlocomotion. These results suggest that the OFC may be critically involved in NMDA receptor-mediated psychotic-like behavioral abnormalities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Most in vivo studies on the conversion to insulin-producing cells with AAV carrying PDX1 gene are performed in rodents. However, there is little information regarding Adeno-associated virus (AAV) carrying PDX1 gene transduced to human liver in vivo because accidental death caused by unpredicted factors cannot be denied, such as the hypoglycemic agent troglitazone with hepatic failure. Here we aim to confirm insulin secretion from human liver transduced with AAV carrying PDX1 gene in vivo and any secondary effect using a humanized liver mouse. As the results, AAV2-PG succeeded to improve the hyperglycemia of STZ-induced diabetic humanized liver mice. Then, the analysis of humanized liver mice revealed that the AAV2-PG was more transducible to humanized liver area than to mouse liver area. In conclusion, the humanized liver mouse model could be used to examine AAV transduction of human hepatocytes in vivo and better predict clinical transduction efficiency than nonhumanized mice., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We examined the pancreatic function of p13 encoded by 1110001J03Rik, whose expression is decreased in pancreatic islets in high-fat-fed diabetic mice, by generating transgenic mice overexpressing p13 (p13-Tg) in pancreatic β-cells. p13-Tg mice showed normal basal glucose metabolism; however, under high-fat feeding, these animals showed augmented glucose-induced first-phase and total insulin secretion, improved glucose disposal, greater islet area and increased mitotic insulin-positive cells. In addition, high-fat diet-induced 4-hydroxynonenal immunoreactivity, a reliable marker and causative agent of lipid peroxidative stress, was significantly decreased in p13-Tg mouse islets. These results indicate that p13 is a novel pancreatic factor exerting multiple beneficial effects against type 2 diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains., (Copyright © 2015 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) play a pivotal role during embryonic development and are required for proper organogenesis, including hematopoiesis. Recent studies suggest that, in the early mesoderm, there is an interaction between the hematopoietic and cardiac lineages. However, whether miRNAs can affect other lineages remains unknown. Therefore, we investigated whether hematopoietic miR-142-3p modulated the mesoderm formation. We report that knockdown (KD) of miR-142-3p, a hematopoietic-specific miRNA, in zebrafish resulted in loss of hematopoiesis during embryonic development. Intriguingly, we observed abnormal cardiac phenotypes and insufficiency of somitegenesis in KD-morphants. In the early developmental stage, a tiny heart, contractile dysfunction in the ventricle, cardiac arrhythmia (e.g. a 2:1 ratio of atrial:ventricular beating), and bradycardia were consistently observed. Histological examination revealed severe hypoplasia of the ventricle and disrupted muscle alignment. To determine the mechanism, we performed DNA microarray analysis. The results revealed that the expression of several mesodermal genes essential for the formation of cardiac and somatic mesoderm, such as no tail, T-box gene 16, mesoderm posterior a, one eye pinhead, and rho-associated, coiled-coil containing protein kinase (Rock2a), were increased in miR-142-3p KD-morphants. The luciferase reporter assay revealed that miR-142-3p repressed luciferase activity on the Rock2a 3'-UTR. The findings of the present study indicate that miR-142-3p plays a critical role in hematopoiesis, cardiogenesis, and somitegenesis in the early stage of mesoderm formation via regulation of Rock2a., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is growing evidence that stromal fibroblasts can promote tumor progression via several mechanisms. We previously reported that podoplanin (PDPN) expressed on stromal fibroblasts is functionally protein responsible for the promotion of tumor formation in mouse subcutaneous tissue. The purpose of the present study was to reveal the molecular mechanism by which PDPN on stromal fibroblasts promotes tumor formation. The subcutaneous co-injection of the human lung adenocarcinoma cell line A549 and human fibroblasts (hFbs) overexpressing wild-type podoplanin (WT-PDPN) promoted subcutaneous tumor formation, compared with the co-injection of A549 and control hFbs (64% vs 21%). On the other hand, hFbs expressing PDPN mutant in which the cytoplasmic domain of PDPN was deleted (PDPN-Del.IC), resulted in a relatively lower level of tumor formation (33%). Since PDPN reportedly regulates RhoA activity through its cytoplasmic domain, we measured the activation state of RhoA in hFbs expressing WT-PDPN. RhoA activity was 2.7-fold higher in WT-PDPN expressing hFbs than in control hFbs. Furthermore, the subcutaneous co-injection of hFbs expressing constitutive active RhoA (G14VRhoA) and A549 cells enhanced tumor formation compared with the co-injection of the same cell line and control hFbs. These results indicate that enhanced RhoA activity in hFbs expressing PDPN may be one of the mechanisms resulting in the promotion of tumor formation, suggesting that biomechanical remodeling of the microenvironment by stromal fibroblasts may play important roles in tumor progression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice display remarkable behavioral changes including increased novelty-seeking behavior and reduced hypothermia induced by either serotonin (5-HT)(1A) receptor agonists or ethanol. Because 5-HT(1A) receptors have been implicated in the development of alcohol dependence, we have examined ethanol preference in PACAP-deficient mice using a two-bottle choice and a conditioned place preference test, as well as additive effects of ethanol and 5-HT(1A) receptor agents on hypothermia. PACAP-deficient mice showed an increased preference towards ethanol compared with wild-type mice. However, they showed no preference for the ethanol compartment after conditioning and neither preference nor aversion to sucrose or quinine. The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) restored the attenuated hypothermic response to ethanol in the mutants to similar levels in wild-type mice, with no effect in wild-types. In contrast, the 5-HT(1A) receptor antagonist WAY-100635 attenuated the ethanol-induced hypothermia in wild-type mice, with no effect in the mutants. These results demonstrate increased ethanol preference in PACAP-deficient mice that may be mediated by 5-HT(1A) receptor-dependent attenuation of ethanol-induced central inhibition., (Copyright 2009 Elsevier Inc. All rights reserved.)

Prebeta1-HDL, a putative discoid-shaped high-density lipoprotein (HDL) is known to participate in the retrieval of cholesterol from peripheral tissues. In this study, to clarify potential sources of this lipoprotein, we conducted heparin injection on four Japanese volunteer men and found that serum triglyceride (TG) level decreased in parallel with the increase in serum nonesterified fatty acids and plasma lipoprotein lipase (LPL) protein mass after heparin injection. Plasma prebeta1-HDL showed considerable increases at 15 min after the heparin injection in all of the subjects. In contrast, serum HDL-C levels did not change. Gel filtration with fast protein liquid chromatography system (FPLC) study on lipoprotein profile revealed that in post-heparin plasma, low-density lipoprotein and alphaHDL fractions did not change, whereas there was a considerable decrease in very low-density lipoprotein (VLDL) fraction and an increase in prebeta1-HDL fraction when compared with those in pre-heparin plasma. We also conducted in vitro analysis on whether prebeta1-HDL was produced during VLDL lipolysis by LPL. One hundred microliters of VLDL extracted from pooled serum by ultracentrifugation was incubated with purified bovine milk LPL at 37 degrees C for 0-120 min. Prebeta1-HDL concentration increased in a dose dependent manner with increased concentration of added LPL in the reaction mixture and with increased incubation time, indicating that prebeta1-HDL was produced during lipolysis of VLDL by LPL. Taken these in vivo and in vitro analysis together, we suggest that lipolysis of VLDL particle by LPL is an important source for formation of prebeta1-HDL.

We previously reported that immature starfish oocytes contain a novel 530-kDa proteasome-associating complex PC500 [previously named PC530; E. Tanaka, M. Takagi Sawada, C. Morinaga, H. Yokosawa, H. Sawada, Isolation and characterization of a novel 530-kDa protein complex (PC 530) capable of associating with the 20S proteasome from star fish oocytes, Arch. Biochem. Biophys. 374 (2000) 181-188]. In the present study, in order to obtain an insight into the biological function of this complex, we investigated the effects of anti-PC500 monoclonal antibodies on oocyte maturation of the starfish Asterina pectinifera. A monoclonal antibody 7C5 strongly inhibited germinal vesicle breakdown (GVBD) in a concentration-dependent manner. In contrast to the inhibitory effect of the 7C5 antibody on GVBD, no inhibition of egg cleavage was observed in a 7C5-antibody-microinjected single blastomere in a 2-cell stage embryo. These results indicate that PC500 plays a key role in starfish oocyte maturation in a meiosis-specific manner.

There has been much focus recently on the possible functions of apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, in cardiovascular and central nervous systems. We report a new function of apelin as a novel angiogenic factor in retinal endothelial cells. The retinal endothelial cell line RF/6A highly expressed both apelin and APJ transcripts, while human umbilical venous endothelial cells (HUVECs) only expressed apelin mRNA. In accordance with these observations, apelin at concentrations of 1 pM-1 microM significantly enhanced migration, proliferation, and capillary-like tube formation of RF/6A cells, but not those of HUVECs, whereas VEGF stimulates those parameters of both cell types. In vivo Matrigel plug assay for angiogenesis, the inclusion of 1 nM apelin in the Matrigel resulted in clear capillary-like formations with an increase of hemoglobin content in the plug. This is the first report showing that apelin is an angiogenic factor in retinal endothelial cells.

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is one of the neurotransmitters that transfers light signals from the retina to the hypothalamic suprachiasmatic nucleus (SCN) where the master clock of mammalian circadian rhythm locates, and is suggested to be implicated in the mechanism of light-induced phase shift of the circadian clock. Here, we examined changes in the phase shift of circadian rhythm in behavioral activity in mice lacking PACAP (PACAP(-/-)). The phase advance in PACAP(-/-) mice by a light stimulation at late subjective night was significantly attenuated, but the phase delay due to the illumination at the early subjective night slightly diminished. In contrast, the induction of c-Fos in the SCN by the illumination at the early subjective night but not that at the late subjective night was significantly blunted in PACAP(-/-) mice. These data provide new aspects about the roles of PACAP in light-induced phase shift of the circadian clock.

Neuropeptides usually exert a long-lived modulatory effect on the small-molecule neurotransmitters with which they colocalize via regulation of the response times of second messenger systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuromodulator and neurotransmitter and regulates a variety of physiological processes. PACAP is structurally highly conserved during evolution, implying its vital importance. In Drosophila, loss-of-function mutations in a PACAP-like neuropeptide gene, amnesiac (amn), affect both memory retention and ethanol sensitivity. The amnesiac gene is expressed in neurons innervating the mushroom body lobes, the olfactory associative learning center. Conditional genetic ablation of neurotransmitter release from these neurons mimics the amnesiac memory phenotypes, suggesting an acute role for amnesiac in memory. However, genetic rescue experiments also suggest developmental defects in amnesiac mutants, implying a role in neuronal development. There is a parallel between memory formation in Drosophila and mammals. PACAP-specific (PAC(1)) receptor-deficient mice show a deficit in hippocampus-dependent associative learning and mossy fiber long-term potentiation (LTP). Meanwhile, PACAP-deficient mice display a high early mortality rate and additional CNS phenotypes including behavioral and psychological phenotypes (e.g., hyperlocomotion, intense novelty-seeking behavior, and explosive jumping). A functional comparison between PACAP and amnesiac underlines phylogenetically conserved functions across phyla and may provide insights into the possible mechanisms of action and evolution of this neuropeptidergic system.

We report the isolation and characterization of a cDNA coding for Fugu rubripes prion protein (PrP)-like of 180 amino acids which includes the PrP-conserved hydrophobic region homologous to that of Xenopus PrP. In addition to the hydrophobic region, Fugu PrP-like has several features common to PrPs, such as a signal sequence, a basic nature (pI 9.7) and a single intron in the 5' untranslated region. A possible glycosyl phosphatidylinositol (GPI) anchor site also exists in PrP-like. In expression analysis, PrP-like mRNA was detected in retina, skin, and brain, all of which express PrP mRNA in mammals. In a genome fragment clone (T002589, 31945 bp) sequenced by the Fugu Genomics Project, PrP-like located between KIAA0168 and SLC231A homologues. In human chromosome 20p13, PrP, Doppel, KIAA0168, and SLC231A align in this order. The close gene arrangement between the Fugu and human genomes suggests that Fugu PrP-like is a real orthologue of human PrP. However, Fugu PrP-like does not possess tandem repeats or a region with two glycosylation sites and a disulphide bridge. We do not declare that the cloned Fugu PrP-like represents fish PrP due to structural inconsistency, but believe that it will offer new insights into the evolution of PrPs from fish to tetrapods., ((c) 2002 Elsevier Science (USA).)

We have recently shown that in PC12 cells, PACAP and NGF synergistically increase PACAP gene transcription and mRNA level, and that the MAPK/ERK kinase inhibitor PD98059 blocks the PACAP mRNA expression induced by either PACAP or NGF, but not that induced by the combination, suggesting involvement of multiple signaling pathways. Here we show that the p38 MAPK inhibitor SB203580 almost completely inhibits the PACAP mRNA expression induced by PACAP alone or in combination with NGF. PACAP induces neurite outgrowth and potentiates NGF-induced neurite outgrowth in PC12h cells. Unlike the case for the PACAP mRNA expression, SB203580 did not affect, but PD98059 reduced, PACAP and NGF-induced neurite outgrowth. These results indicate that PACAP receptors are coupled to the p38 signaling pathway, and that p38 plays a key role in the regulation of PACAP gene expression, while ERK, but not p38, MAPK is involved in PACAP and NGF-induced neurite outgrowth., (Copyright 2001 Academic Press.)

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE., (Copyright 2000 Academic Press.)

Three type II membrane proteins Anp1, Van1 and Mnn9 of Saccharomyces cerevisiae share significant sequence homology. Their precise biochemical activity has long been unknown though the mutant phenotype indicates their participation in protein glycosylation in the Golgi apparatus. To shed light on their molecular characteristics, interactions of these proteins were studied by immunoprecipitation after solubilizing the membrane by nonionic detergent. Our results indicated that there are at least two submembrane complexes containing these proteins: one contains Van1 and Mnn9 proteins and the other contains Anp1 and Mnn9 proteins. In addition, Hoc1 protein which has significant homology to Och1 protein colocalized with Anp1 and Mnn9 proteins. These complexes with similar but partially different constituents may represent essential parts of glycosylation machinery in the yeast Golgi compartments.

Transfer of the gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis in scurvy-prone animals, into medaka (Oryzias latipes) was successfully done. The expression plasmid pSVL-GLO, carrying rat liver L-gulono-gamma-lactone oxidase cDNA, was microinjected into the cytoplasm of fertilized eggs during the one-cell stage. Four male F0 fish having the transgene in their germ cells came to maturity, and F1 progeny derived from one of the F0 fish possessed L-gulono-gamma-lactone oxidase activity, indicating that the transgene was functionally expressed in the fish. Genomic Southern blot analysis demonstrated that the transgene existed in both chromosome-integrated and extrachromosomal forms.

Heterogeneity of the gamma-subunit of G proteins has been demonstrated by cDNA cloning and by partial sequence analyses. We have isolated two intact beta gamma-subunit isoforms from bovine brain Gi/o mixture, in which only gamma subunits are distinct (Sohma, H., et al. (1992) Biochem. Biophys. Res. Commun. 184, 175-182). In this study, we isolated the gamma-subunit isoforms, gamma-I and gamma-II, and examined their amino acid sequences. Both gamma-I and gamma-II had blocked N-terminal amino acid residues, and the terminal amino acids of both were able to be truncated by an acylamino-acid-releasing enzyme. Gamma-I seemed to be identical with the gamma-subunit reported elsewhere, while the gamma-II appeared to be a novel protein. Antibodies to synthetic peptides based on the part of the amino acid sequences of gamma-I and gamma-II reacted specifically to gamma-I and gamma-II, respectively.

When a mixture of bovine brain G-proteins (Gi/o) was loaded onto an octyl sepharose column in the presence of AlF4-, alpha-subunits of molecular weights 39 kDa and the 41 kDa were eluted separately, followed by the appearance of two distinct peaks containing beta gamma-subunits (beta gamma-I, beta gamma-II). Both beta gamma-I and beta gamma-II possessed identical beta-subunits but different gamma-subunits. The molecular weights of the two gamma-subunits determined by SDS-polyacrylamide gel electrophoresis both in the presence and absence of urea were 4.5 kDa (gamma-I) and 5.0 kDa (gamma-II). Tests indicated that the two isolated gamma-subunits are intact and have not undergone proteolysis. The amino acid composition of gamma-I appeared to be distinct from that of gamma-II. Therefore, this method is a simple procedure for isolating beta gamma-I and beta gamma-II.

Previously, we reported a case with type I hyperlipidemia due to a lipid interface recognition deficiency in lipoprotein lipase (LPL) (1). The LPL from postheparin plasma of this patient did not hydrolyze TritonX-100-triolein or very low density lipoprotein-triolein but did hydrolyze tributyrin and LysoPC-triolein substrates. Sequence analysis of the probands DNA revealed a heterozygous nucleotide change: a C----G transversion at position of 1595, resulting in changing the codon for Ser447 to a stop codon. Expression studies of this mutant LPLcDNA in Cos-1 cells produced and secreted considerable amounts of LPL mass in the culture media. The mutated LPL hydrolyzed much less TritonX-100-triolein than wild type LPL, whereas hydrolysis of tributyrin and LysoPC--triolein was the same with both the mutant and wild type LPL. These results suggest that this mutation might be responsible for the property of the LPL with a defect in lipid interface recognition in the type I patient we reported.

Thrombin induced an increase in [Ca2+]i in mouse mastocytoma P-815 cells. This increase was markedly reduced by prior exposure to pertussis toxin (PT) but not by removal of extracellular Ca2+, suggesting that thrombin stimulates phospholipase C via a PT-sensitive GTP-binding protein. ATP also induced an increase in [Ca2+]i. This increase was insensitive to PT but completely suppressed on removal of extracellular Ca2+, suggesting that ATP stimulates Ca2+ influx in a PT-insensitive manner. Iloprost, a stable prostacyclin analogue, increased the cellular cAMP level and dose-dependently inhibited the thrombin-induced increase in [Ca2+]i, whereas the ATP-induced increase in [Ca2+]i was markedly enhanced by iloprost. Cyclic AMP analogues, dibutyryl cAMP and 8-bromo cAMP, also inhibited the increase in [Ca2+]i induced by thrombin and promoted that by ATP, indicating that the inhibitory and stimulatory effects of iloprost are mediated by cAMP. These results suggest that the prostacyclin receptor differentially regulates two distinct Ca2+ mobilizing systems via cAMP in mastocytoma cells.

Treatment of a human promyelocytic leukemia cell line (HL-60) with etoposide for 3-4 hrs produced an extensive degradation of DNA. Agarose gel electrophoresis showed DNA fragmentation in a nucleosomal ladder pattern. Simultaneous addition of zinc ion (ZnSO4, 1 mM) inhibited DNA fragmentation, although the amount of DNA strand breakage introduced initially by etoposide did not change significantly as measured by the DNA unwinding assay. Furthermore, zinc ion abrogated both the activation of poly(ADP-ribose) synthesis and the morphologic changes characteristic of apoptosis by etoposide. These results suggest that zinc ion inhibits a metabolic process somewhere between initial DNA cleavage through an interference with type II topoisomerase and delayed degradation of cellular DNA to a nucleosome-like pattern.

Addition of concanavalin A (Con A) to mouse neuroblastoma N18TG2 cells cultured with dibutyryl-cAMP which can stimulate neurite outgrowth, stopped the neuritic processes effectively. The extended neurites showed a gradual retraction for at least 8 hrs after addition of Con A, while addition of colchicine caused rapid retraction of the neurites. Immunocytochemistry showed that the addition of Con A did not disorganize the microtubules but the addition of colchicine did. The increase in beta-tubulin mRNA expression which was observed after cell culture and after stimulation by dB-cAMP was suppressed by the addition of Con A. Con A did not affect the beta-tubulin mRNA expression when the cells had already been cultured, while colchicine drastically decreased it. Thus, Con A appeared to affect the beta-tubulin mRNA expression in a different manner from colchicine, probably through inhibition of cell movement.