Risk Factors for Acetaminophen-induced Liver Injury: A Single-center Study from Japan.

PURPOSE: Acetaminophen has been increasingly used for the treatment of cancer-related pain in Japan since the revision of the package insert on January 21, 2011. However, high-dose acetaminophen may cause liver injury. The objectives of this study were to investigate the prevalence of liver injury in patients receiving acetaminophen and to identify the risk factors. METHODS: The subjects were patients who were treated with acetaminophen ≥1500 mg/d for ≥4 weeks at Ehime University Hospital between April 2011 and December 2014. Drug-induced liver injury was evaluated by alanine aminotransferase and alkaline phosphatase levels, Naranjo score, and Child-Pugh classification. FINDINGS: A total of 287 of 562 patients were treated for 4 weeks with acetaminophen ≥1500 mg/d. Twenty of 102 patients analyzed had drug-induced liver injury. Multivariate analysis was performed with variables from the results of univariate analysis (sex, age ≥70 years, abnormal alanine aminotransferase and alkaline phosphatase levels, and serious liver disease), and age ≥70 years and serious liver disease were significant risk factors. IMPLICATIONS: The findings from the present observational, single-center study suggest that serious liver disease before administration is an independent risk factor for acetaminophen-induced liver injury.

Mutations induced by 8-oxo-7,8-dihydroguanine in WRN- and DNA polymerase λ-double knockdown cells.

Reactive oxygen species generate 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine), which induces G:C→T:A transversion mutations. The knockdowns of the protein responsible for Werner syndrome (WRN), a cancer-associated DNA helicase, and DNA polymerase (pol) λ, a WRN-interacting DNA pol, cause untargeted base-substitution mutations (action-at-a-distance mutations). To examine the consequences of the dual reductions of WRN and pol λ for the mutations caused by GO, siRNAs against both proteins were introduced into human U2OS cells. A replicable plasmid DNA with the oxidised nucleobase in a unique position of the supF gene was then introduced into the double knockdown cells. The amplified DNA recovered from the cells was used to transform a bacterial indicator strain. The mutant frequency and the subsequent sequence analysis revealed that the double knockdown additively promoted the G:C→T:A substitution at the GO position and increased the action-at-a-distance mutations to a level similar to that of the single WRN knockdown. Thus, WRN and DNA pol λ seem to suppress the targeted G:C→T:A mutation at least in part independently and reduce the untargeted mutations via an identical pathway.

Decreased parvalbumin and somatostatin neurons in medial prefrontal cortex in BRINP1-KO mice.

BRINPs (BMP/RA-inducible Neural Specific Protein-1, 2, 3) are family genes expressed mainly in both the central and peripheral nervous system. BRINP1 is abundantly expressed in many of adult brain regions including cerebral cortex and hippocampus, with expression regulated in an activity-dependent manner in the dentate gyrus. Mice with disrupted BRINP1 gene exhibit abnormal behaviors such as increased locomotive activity and poor social activity which are analogous to symptoms of human psychiatric disorders such as schizophrenia (SCZ), autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). In the present study, to clarify the physiological roles of BRINP1 in psychiatric disorders, we examined the numbers of parvalbumin (PV)-expressing neurons and somatostatin (SST)-expressing neurons in the medial prefrontal cortex (mPFC) in BRINP1-KO mice. Immunohistochemical analysis revealed the numbers of PV-expressing neurons and SST-expressing neurons in mPFC of BRINP1-KO mice were, respectively, 50% and 20% fewer than corresponding neurons in mPFC of wild-type mice. These data suggest that the abnormal behaviors related to human psychiatric disorders in BRINP1-KO mice could be derived from the hyperexcitability of pyramidal neurons as a consequence of decreased inhibitory innervation and conceivable dysregulation of the Excitatory/Inhibitory balance in mPFC.

Existence of NEU1 sialidase on mouse thymocytes whose natural substrate is CD5.

Membrane-bound sialidases in the mouse thymus are unique and mysterious because their activity at pH 6.5 is equal to or higher than that in the acidic region. The pH curve like this has never been reported in membrane-bound form. To clarify this enzyme, we studied the sialidase activities of crude membrane fractions from immature-T, mature-T and non-T cells from C57BL/6 mice and from SM/J mice, a strain with a defect in NEU1 activity. Non-T cells from C57BL/6 mice had high activity at pH 6.5, but those from SM/J mice did not. Neu1 and Neu3 mRNA was shown by real-time PCR to be expressed in T cells and also in non-T cells, whereas Neu2 was expressed mainly in non-T cells and Neu4 was scarcely expressed. However, the in situ hybridization study on the localization of four sialidases in the thymus showed that Neu4 was clearly expressed. We then focused on a sialidase on the thymocyte surface because the possibility of the existence of a sialidase on thymocytes was suggested by peanut agglutinin (PNA) staining after incubation of the cells alone in PBS. This activity was inhibited by NEU1-selective sialidase inhibitor C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The natural substrate for the cell surface sialidase was identified as clustered differentiation 5 (CD5) by PNA-blot analysis of anti-CD5 immunoprecipitate. We conclude that NEU1 exists on the cell surface of mouse thymocytes and CD5 is a natural substrate for it. Although this is not the main reaction of the membrane-bound thymus-sialidases, it must be important for the thymus.

Psychiatric Patients with Antipsychotic Drug-Induced Hyperprolactinemia and Menstruation Disorders.

Treatment with antipsychotic drugs has been associated with hyperprolactinemia. The same antipsychotic drugs have also been associated with side effects such as menstruation disorders. The aim of this study was to evaluate the prevalence of hyperprolactinemia and menstruation disorders in women undergoing antipsychotic treatment. We performed a retrospective chart review study of psychiatric patients who underwent laboratory testing for serum prolactin (PRL) level between March 2011 and March 2015 in Ehime University Hospital. Patients presenting with and without menstruation disorders were evaluated to determine if they presented concomitant hyperprolactinemia. Patients with menstrual disorders had a significant increase in serum PRL level with a mean of approximately 90 ng/mL. Those with menstrual disorders presented increased PRL levels by 2-fold that of patients without menstrual disorder. However, there was no significant difference in the equivalent dose of chlorpromazine between these two groups. Additionally, about 70% of patients with menstrual disorders received risperidone treatment. The receiver operating characteristic curve showed that the optimal cutoff point of serum PRL level associated with the development of menstrual disorders was 60 ng/mL. Based on these results, we concluded that patients with menstrual disorders presented increased serum PRL, and that most of them underwent treatment with risperidone.

Interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1): Implication of N-glycosylation in FAM5C secretion.

N-glycosylation of proteins is important for protein folding and function. We have recently reported that FAM5C/BRINP3 contributes to the tumor necrosis factor-α-induced expression of leukocyte adhesion molecules in vascular endothelial cells (ECs). However, regulatory mechanism of the FAM5C biosynthesis is poorly understood. Co-immunoprecipitation assay revealed the interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a glycoprotein folding-sensor enzyme. FAM5C ectopically expressed in HEK293 cells was localized to the endoplasmic reticulum and co-localized with endogenously expressed UGGT1. Molecular size of FAM5C was reduced by treatment with N-glycosidase F and in FAM5C-expressing cells cultured in the presence of the N-glycosylation inhibitor tunicamycin. FAM5C was secreted by the cells and the secretion of FAM5C was blocked by tunicamycin. Among six potential N-glycosylation sites, the potential site at Asn168 was not N-glycosylated, and Asn337, Asn456, Asn562, Asn609, and Asn641 mutants were poorly secreted by the cells. These results demonstrated that FAM5C is an N-glycosylated protein and N-glycosylation is necessary for the secretion of FAM5C.

Cleavage of Target DNA Promotes Sequence Conversion with a Tailed Duplex.

Base sequence conversion in target DNA is achieved when a 5'-tailed duplex (TD) is introduced into cells. In this study, the effects of target DNA cleavage on sequence conversion with a TD were examined. Plasmid DNAs with and without cleavage near the target position were each introduced into HeLa cells, together with the TD. The cleavage promoted the sequence alteration efficiency by ca. 7-fold. These results suggested that the sequence conversion efficiency with the TD fragment is increased when an artificial nuclease introduces cleavage near the target site.

Action-at-a-distance mutagenesis induced by oxidized guanine in Werner syndrome protein-reduced human cells.

8-Oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA is one of the most important oxidatively damaged bases and causes G:C → T:A substitution mutations. The Werner syndrome protein (WRN) is a cancer-related RecQ DNA helicase and plays many roles in DNA replication and repair. To examine the relationships between G(O)-induced mutations and WRN, shuttle plasmid DNA containing a G(O):C pair in the supF gene was transfected into human U2OS cells, in which WRN was knocked down. The plasmid DNA replicated in the knockdown cells was introduced into an Escherichia coli indicator strain. The knockdown of WRN increased the mutant frequency of the G(O)-plasmid DNA. Unexpectedly, however, the WRN knockdown only slightly enhanced the targeted G:C → T:A mutation. Instead, base-substitution mutations at various positions were more frequently detected, with statistical significance. The results obtained in this study suggested that the reduction of the cancer-related WRN induced action-at-a-distance mutagenesis by the G(O):C pair in human cells. In addition, the WRN knockdown decreased the G(O):A-induced A:T → C:G mutations, suggesting that WRN may enhance the mutations caused by G(O) in the nucleotide pool.

Induction of action-at-a-distance mutagenesis by 8-oxo-7,8-dihydroguanine in DNA pol λ-knockdown cells.

INTRODUCTION: In DNA, 8-oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) is one of the most pivotal oxidatively damaged bases and induces G:C → T:A transversion mutations. DNA polymerase λ preferentially inserts dCTP opposite G(O) in vitro, and this error-free bypass function is considered to be important after A base removal from G(O):A pairs by the MUTYH DNA glycosylase. To examine the effects of reduced levels of DNA polymerase λ on the G(O)-induced mutations, the polymerase was knocked-down in human U2OS cells, and a shuttle plasmid DNA containing a G(O):C pair at position 122 in the supF gene was transfected into the cells. The plasmid DNA replicated in the cells was introduced into an Escherichia coli indicator strain, to measure the supF mutant frequency. RESULTS: The knockdown of DNA polymerase λ significantly enhanced the mutant frequency of the G(O) plasmid DNA. Contrary to our expectations, the knockdown did not promote the targeted G:C → T:A transversion. Instead, substitution mutations at G:C sites other than position 122 were enhanced in the cells. CONCLUSIONS: These results suggested that the knockdown of DNA polymerase λ induced action-at-a-distance mutagenesis in human cells when the G(O):C pair was present in the DNA.

Absence of BRINP1 in mice causes increase of hippocampal neurogenesis and behavioral alterations relevant to human psychiatric disorders.

BACKGROUND: We have previously identified BRINP (BMP/RA-inducible neural-specific protein-1, 2, 3) family genes that possess the ability to suppress cell cycle progression in neural stem cells. Of the three family members, BRINP1 is the most highly expressed in various brain regions, including the hippocampus, in adult mice and its expression in dentate gyrus (DG) is markedly induced by neural activity. In the present study, we generated BRINP1-deficient (KO) mice to clarify the physiological functions of BRINP1 in the nervous system. RESULTS: Neurogenesis in the subgranular zone of dentate gyrus was increased in BRINP1-KO mice creating a more immature neuronal population in granule cell layer. The number of parvalbumin expressing interneuron in hippocampal CA1 subregion was also increased in BRINP1-KO mice. Furthermore, BRINP1-KO mice showed abnormal behaviors with increase in locomotor activity, reduced anxiety-like behavior, poor social interaction, and slight impairment of working memory, all of which resemble symptoms of human psychiatric disorders such as schizophrenia and attention-deficit/hyperactivity disorder (ADHD). CONCLUSIONS: Absence of BRINP1 causes deregulation of neurogenesis and impairments of neuronal differentiation in adult hippocampal circuitry. Abnormal behaviors comparable to those of human psychiatric disorders such as hyperactivity and poor social behavior were observed in BRINP1-KO mice. These abnormal behaviors could be caused by alteration of hippocampal circuitry as a consequence of the lack of BRINP1.

Anatomy of plasmid DNAs with anti-silencing elements.

The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected.

Enhanced transgene expression from chromatinized plasmid DNA in mouse liver.

Plasmid DNA was chromatinized with core histones (H2A, H2B, H3, and H4) in vitro and was delivered into mouse liver by hydrodynamics-based administration. Transgene expression from the chromatinized plasmid DNA was more efficient than that from plasmid DNA delivered in the naked form. The use of acetylation-enriched histones isolated from cells treated with a histone deacetylase inhibitor (trichostatin A) seemed to be more effective. These results indicated that chromatinized plasmid DNA is useful for efficient transgene expression in vivo.

Analysis of the expression and function of BRINP family genes during neuronal differentiation in mouse embryonic stem cell-derived neural stem cells.

We previously identified a novel family of genes, BRINP1, 2, and 3, that are predominantly and widely expressed in both the central nervous system (CNS) and peripheral nervous system (PNS). In the present study, we analyzed the expression pattern of three BRINP genes during differentiation of mouse embryonic stem (ES) cell-derived neural stem cells (NSCs) and their effects on the cell-cycle regulation of NSCs. While there was no significant expression of any BRINP-mRNA expressed in mouse ES cells, BRINP 1 and 2-mRNAs was expressed at high levels in the ES cell-derived neural stem cells. Upon differentiation into neuronal cells in the presence of retinoic acid and BDNF, all three types of BRINP-mRNA were induced with a similar time course peaking at day three of treatment. Upon differentiation into astroglial cells in the presence of serum, BRINP1-mRNA was slightly up-regulated, while BRINP2- and BRINP3-mRNAs were almost abolished in the astrocytes. While 69.2, 26.1, and 7.7% of cells in a population of NSCs in the exponentially growing phase were in the G1, S and G2 phases, respectively, over-expression of any one of the three BRINP genes completely abolished cells in the G2 phase and significantly reduced the cells in S phase to 11.8-13.8%. Based on these results, the physiological roles of induced BRINP genes in the cell-cycle suppression of terminally differentiated post-mitotic neurons are discussed.

The generation and behavioral analysis of ceramide kinase-null mice, indicating a function in cerebellar Purkinje cells.

The discovery of ceramide kinase (CerK), which phosphorylates ceramide (Cer) to ceramide 1-phisphate (C1P), established a new pathway for Cer metabolism. Among mouse tissues, brain contains the highest CerK activity. In this study, we found that CerK is highly expressed in cerebellar Purkinje cells. Since Purkinje cells are important for motor-related behaviors, we generated CerK-null mice and performed behavioral analyses. The CerK-null mice were healthy, and displayed no histological abnormalities. The mice lost CerK activity completely, suggesting that CerK is the only enzyme that phosphorylate Cer. However, cellular C1P levels were not different between the CerK-null and wild-type mice, indicating the presence of other C1P-producing pathway. The general motor-coordination was not impaired in the CerK-null mice, but emotional behavior was slightly affected. Our findings suggest that CerK is not necessary for survival at an individual level, but might be involved in higher brain function related to emotion.

Identification and expression analysis of rainbow trout pumilio-1 and pumilio-2.

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.

Activity-dependent regulation of BRINP family genes.

We previously identified a family of novel developmentally regulated genes: BRINP1, 2, and 3, which are predominantly and widely expressed in the CNS from earlier developmental stages to adulthood. In the present study, we investigated the activity-dependent regulation of BRINP expression in the CNS. Among the three BRINP genes, BRINP1-mRNA was specifically up-regulated in the dentate gyrus of mouse hippocampus by kainic acid treatment. In cultured hippocampal neurons, the induction of BRINP1-mRNA was also observed by the activation of glutamate receptors. Although BDNF-mRNA is up-regulated in a similar activity-dependent manner, BDNF itself did not induce BRINP1-mRNA. From these results, the physiological roles of the activity-dependent induction of BRINP1-mRNA are discussed.

Bone morphogenetic protein and retinoic acid-inducible neural specific protein-3 is expressed in gonadotrope cell pituitary adenomas and induces proliferation, migration, and invasion.

Pituitary tumors are common intracranial neoplasms that often result in endocrine dysfunction due to hormone overproduction or deficiencies from mass effects. Gonadotrope cell or gonadotropinomas are tumors that produce LH and/or FSH and represent 40% of macroadenomas. Little is known about their underlying pathogenic mechanisms. We compared expression profiles of 10 gonadotropinomas with nine normal pituitaries by cDNA array and identified bone morphogenetic protein- and retinoic acid-inducible neural-specific protein-3 (BRINP3) as overexpressed in tumors, compared with normals. BRINP3 is a novel, normally brain restricted protein of unknown function. BRINP3 mRNA was expressed selectively in gonadotropinomas. Subcellular localization studies showed that BRINP3 was targeted to the mitochondria, but BRINP3 overexpression was unable to protect pituitary cells against programmed cell death induced by growth factor withdrawal. However, BRINP3 overexpression in pituitary gonadotrope cells promoted proliferation, migration, and invasion. A BRINP3 antibody was raised that demonstrated clustered expression of BRINP3 protein in gonadotropinomas and not in normal human pituitary samples. Thus, BRINP3 is a mitochondrially localized protein that is selectively up-regulated in human gonadotropinomas. Its actions to increase proliferation, migration, and invasion suggest it may play an important role in pituitary tumorigenesis.

Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP-induced morphologic changes.

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP-treated and non-treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP-treated astrocytes after stimulation with ATP by high-performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1-43 imaging, appeared in cyclic AMP-treated astrocytes in a punctiform fashion, but not in non-treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP-induced morphologic changes, and in particular, a v-SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP-treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP-induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP-treated astrocytes in response to ATP released from neighboring neurons and astrocytes.

Role of NRSF/REST in the molecular mechanisms regulating neural-specific expression of trkC/neurotrophin-3 receptor gene.

The processes of differentiation and development of neurons involve the induction of neuron-specific genes by instructive signals with subsequent neurotrophic factor-driven survival and functional maturation. We have previously shown that bone morphogenetic protein-2 (BMP2) and retinoic acid synergistically induce the responsiveness of developing sympathetic neurons to neurotrophic factors, neurotrophin 3 (NT-3), and GDNF by upregulating corresponding receptors concomitantly with the induction of other neuron-specific genes including BRINP1, a neuron-specific cell-cycle regulatory protein. In the present study, we analyzed transcriptional mechanisms regulating the neuron-specific expression of TrkC/NT-3 receptor gene. TrkC gene contains at least four NRSE/RE-1 (neuron-restrictive silencing element/repressor element 1)-like elements (TrkC-NRSE A-D). Consequently, we found that in non-neuronal cells, neuron-restrictive silencing factor (NRSF) acts on TrkC-NRSE D located at the downstream of exon 3 to suppress the promoter activity of TrkC gene in a manner similar to the mechanism of NRSF suppressing BRINP1 transcription. In contrast, in neuronal cells, the biological activity of NRSF on TrkC was suppressed. From these observations, molecular mechanisms regulating the expression of neuron-specific genes via NRSE during neuronal differentiation are discussed.

Autocrine action of BMP2 regulates expression of GDNF-mRNA in sciatic Schwann cells.

Schwann cell is a cell type that forms myelin sheath and provides trophic supports for neuronal cells by producing neurotrophic factors in both normal and traumatic situations. It was recently reported that after lesion of sciatic nerve, mRNA for glial cell line-derived neurotrophic factor (GDNF) is induced in nonneuronal cells in the nerve. However, the mechanism regulating GDNF-mRNA has remained largely unknown. In the present study, we searched for factors regulating the GDNF-mRNA expression in Schwann cells. First, we found that after transfer into explant culture as an in vitro lesion model, sciatic nerve segments began to express mRNA for bone morphogenetic protein-2 (BMP2) concomitantly with the induction of GDNF-mRNA. Treatment of the Schwann cells isolated from the sciatic nerve with combination of BMP2 and retinoic acid (RA) dramatically induced GDNF-mRNA, while BMP2 or RA alone had no effect. Furthermore, ionomycin, a calcium ionophore, which had even stronger activity on the induction of GDNF-mRNA also induced also BMP2-mRNA in cultured Schwann cells. Effects of inhibitors of intracellular signaling pathways such as protein kinase C inhibitor and MAPKK inhibitor suggested that the molecular mechanism of the induction of GDNF-mRNA is distinct from that of BMP2-mRNA. These results suggest that the Schwann cell-produced BMP2 plays an important role in the induction of GDNF after nerve injury in an autocrine fashion.