Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of ß-propeller associated neurodegeneration.

ß-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21 and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including ß-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3 and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.

TERT upregulation promotes cell proliferation via degradation of p21 and increases carcinogenic potential.

Telomerase reverse transcriptase (TERT) gene aberration is detectable in >80% of cases with hepatocellular carcinoma (HCC). TERT reactivation is essential for cellular immortalization because it stabilizes telomere length, although the role of TERT in hepatocarcinogenesis remains unelucidated. To elucidate the significance of aberrant TERT expression in hepatocytes in inflammation-associated hepatocarcinogenesis, we generated Alb-Cre;TertTg mice, which overexpress TERT in the liver and examined their phenotype during chronic inflammation. Based on transcriptome data from the liver tissue of Alb-Cre;TertTg mice, we examined the role of TERT in hepatocarcinogenesis in vitro. We also evaluated the relationship between TERT and cell-cycle-related molecules, including p21, in HCC samples. The liver tumor development rate was increased by TERT overexpression during chronic inflammation, especially in the absence of p53 function. Gene set enrichment analysis of liver tissues revealed that gene sets related to TNF-NFκB signaling, cell cycle, and apoptosis were upregulated in Alb-Cre;TertTg liver. A luciferase reporter assay and immunoprecipitation revealed that TERT interacted with NFκB p65 and enhanced NFκB promoter activity. On the other hand, TERT formed protein complexes with p21, cyclin A2, and cyclin E and promoted ubiquitin-mediated degradation of p21, specifically in the G1 phase. In the clinical HCC samples, TERT was highly expressed but p21 was conversely downregulated, and TERT expression was associated with the upregulation of molecules related to the cell cycle. Taken together, the aberrant upregulation of TERT increased NFκB promoter activity and promoted cell cycle progression via p21 ubiquitination, leading to hepatocarcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ablation of oligodendrogenesis in adult mice alters brain microstructure and activity independently of behavioral deficits.

Oligodendrocytes continue to differentiate from their precursor cells even in adulthood, a process that can be modulated by neuronal activity and experience. Previous work has indicated that conditional ablation of oligodendrogenesis in adult mice leads to learning and memory deficits in a range of behavioral tasks. The current study replicated and re-evaluated evidence for a role of oligodendrogenesis in motor learning, using a complex running wheel task. Further, we found that ablating oligodendrogenesis alters brain microstructure (ex vivo MRI) and brain activity (in vivo EEG) independent of experience with the task. This suggests a role for adult oligodendrocyte formation in the maintenance of brain function and indicates that task-independent changes due to oligodendrogenesis ablation need to be considered when interpreting learning and memory deficits in this model.

A novel NODAL variant in a young embolic stroke patient with visceral heterotaxy.

BACKGROUND: Ischemic stroke in young adults can be caused by a variety of etiologies including the monogenic disorders. Visceral heterotaxy is a condition caused by abnormal left-right determinations during embryonic development. We aimed to determine the cause of a young ischemic stroke patient with visceral heterotaxy. CASE PRESENTATION: We performed neurological, radiological, and genetic evaluations in a 17-year-old male patient presenting ischemic stroke and visceral heterotaxy to determine the underlying cause of this rare disease combination. Brain magnetic resonance imaging (MRI) showed evidence of embolic stroke, abdominal computed tomography (CT) showed visceral heterotaxy, and echocardiogram showed cardiac anomaly with right-to-left-shunt (RLS). Whole genome sequencing (WGS) revealed a heterozygous missense variant (NM_018055.5: c.1016 T > C, p.(Met339Val)) in the NODAL gene, which is essential to the determination of the left-right body axis. CONCLUSIONS: Our study highlights the importance of evaluating genetic etiology in young ischemic stroke and the need for stroke risk management in visceral heterotaxy patients with RLS. To the best of our knowledge, we report the first genetically-confirmed case of visceral heterotaxy with young embolic stroke reported to date.

On the origin of gastric tumours: analysis of a case with intramucosal gastric carcinoma and oxyntic gland adenoma.

Most gastric cancers develop in inflamed gastric mucosa due to Helicobacter pylori infection, typically with metaplastic changes. However, the origins of gastric cancer remain unknown. Here, we present a case of intramucosal gastric carcinoma (IGC) and oxyntic gland adenoma (OGA) derived from spasmolytic polypeptide-expressing metaplasia (SPEM). Early gastric cancer adjacent to a polyp was found in the upper corpus of a 71-year-old woman without H. pylori infection and was endoscopically resected. Histological examination showed IGC and OGA, both of which had predominant MUC6 expression. Interestingly, gastric glands with enriched MUC6-positive mucous cells, referred to as SPEM, expanded between them. Whole-exome sequencing analysis revealed a truncating KRAS(G12D) mutation in IGC, OGA, and SPEM. In addition, TP53 and CDKN2A mutations and a loss of chromosome 17p were found in the IGC, whereas a GNAS mutation was observed in the OGA. These results indicated that IGC and OGA originated from the KRAS-mutated SPEM. © 2023 The Pathological Society of Great Britain and Ireland.

Brain carbon monoxide can suppress the rat micturition reflex through brain γ-aminobutyric acid receptors.

OBJECTIVES: To investigate roles of brain carbon monoxide (CO), an endogenous gasotransmitter, in regulation of the rat micturition reflex. METHODS: In urethane-anesthetized (0.8 g/kg, ip) male rats, evaluation of urodynamic parameters was started 1 h before intracerebroventricular administration of CORM-3 (CO donor) or ZnPP (non-selective inhibitor of heme oxygenase, a CO producing enzyme) and continued for 2 h after the administration. We also investigated effects of centrally pretreated SR95531 (GABAA receptor antagonist) or SCH50911 (GABAB receptor antagonist) on the CORM-3-induced response. RESULTS: CORM-3 significantly prolonged intercontraction intervals (ICIs) without changing maximal voiding pressure (MVP), while ZnPP significantly shortened ICI and reduced single-voided volume and bladder capacity without affecting MVP, post-voided residual volume, or voiding efficiency. The ZnPP-induced ICI shortening was reversed by CORM-3. The CORM-3-induced ICI prolongation was significantly attenuated by centrally pretreated SR95531 or SCH50911, respectively. CONCLUSIONS: Brain CO can suppress the rat micturition reflex through brain γ-aminobutyric acid (GABA) receptors.

Treatment strategy for cervical lymph node metastases from early-stage tongue and floor of the mouth squamous cell carcinoma using tumour budding and depth of invasion as predictors.

OBJECTIVES: This study aimed to determine whether elective neck dissection can help improve outcomes in early-stage tongue and floor squamous cell carcinoma (SCC) by statistically analysing the relationship between information obtained from biopsy specimens and the incidence and prognosis of cervical lymph node metastasis (CLM). MATERIALS AND METHODS: Biopsy specimens of 103 patients diagnosed with early cT1-T2 cancer of the tongue and floor of the mouth were included. RESULTS: Multivariate analysis showed that the three parameters significantly correlated with CLM, and univariate analyses showed that budding score (BS) ≥ 5 and pathological depth of invasion (pDOI) ≥ 5 mm were independent risk factors for CLM. There were significant differences in the 5-year cumulative disease-specific survival between the BS < 5 and BS ≥ 5 groups, the pDOI < 5 mm and pDOI ≥ 5 mm groups, and the positive and negative budding and depth of invasion (BD) score groups. CONCLUSION: In early-stage tongue and floor of the mouth cancers with maximum tumour diameter ≤ 20 mm, it may be necessary to treat occult CLM during initial surgery based on the following preoperative criteria: pDOI ≥ 5 mm or BS ≥ 5 in biopsy specimens and DOI ≥ 8 mm on imaging. The BD model exhibited the highest specificity and proved helpful for CLM prediction. CLINICAL RELEVANCE: pDOI ≥ 5 mm and BS ≥ 5 were independent predictors of CLM and prognosis in early-stage tongue and floor of the mouth cancers with a maximum tumour diameter of 20 mm.

Life-long oligodendrocyte development and plasticity.

Oligodendrocyte precursor cells (OPCs) originate in localized germinal zones in the embryonic neural tube, then migrate and proliferate to populate the entire central nervous system, both white and gray matter. They divide and generate myelinating oligodendrocytes (OLs) throughout postnatal and adult life. OPCs express NG2 and platelet-derived growth factor receptor alpha subunit (PDGFRα), two functionally important cell surface proteins, which are also widely used as markers for OPCs. The proliferation of OPCs, their terminal differentiation into OLs, survival of new OLs, and myelin synthesis are orchestrated by signals in the local microenvironment. We discuss advances in our mechanistic understanding of paracrine effects, including those mediated through PDGFRα and neuronal activity-dependent signals such as those mediated through AMPA receptors in OL survival and myelination. Finally, we review recent studies supporting the role of new OL production and "adaptive myelination" in specific behaviours and cognitive processes contributing to learning and long-term memory formation. Our article is not intended to be comprehensive but reflects the authors' past and present interests.

Activation of mouse Otop3 proton channels by Zn2.

Otopetrins (Otop1-Otop3) belong to a newly identified family of proton (H+) channels activated by extracellular acidification. Here, we found that Zn2+ activates the mouse Otop3 (mOtop3) proton channels by using electrophysiological patch-clamp techniques. In mOtop3-expressing human embryonic kidney HEK293T cells, a biphasic inward mOtop3 H+ current comprising a fast transient current followed by a sustained current was observed upon extracellular acidification at pH 5.0. No significant activation of the mOtop3 channel was observed at pH 6.5 and 7.4, but interestingly, Zn2+ dose-dependently induced a sustained activation of mOtop3 under these pH conditions. Increasing the Zn2+ concentration had no effect on the reversal potential of the channel currents, suggesting that Zn2+ does not permeate through the mOtop3. The activation of the mOtop3 channel was specific to Zn2+ among divalent metal cations. Our findings reveal a novel modulatory mechanism of mOtop3 proton channels by Zn2+.

Cloning, DNA sequence, and expression of flagellins from high and low virulence strains of Edwardsiella tarda and their macrophage-stimulating activities.

Edwardsiella tarda is a causative pathogen of edwardsiellosis in fish. Our previous studies on high (NUF251) and low (NUF194) virulent strains of E. tarda demonstrated that NUF251 strain induced significantly higher levels of NO and TNF-α from fish and mouse macrophages than NUF194 strain. Subsequent studies suggested that a flagellin-like protein secreted from E. tarda might be a responsible factor for the macrophage-stimulating activities. To evaluate the activities of flagellins of E. tarda, in this study, the flagellin genes of NUF251 and NUF194 strains were isolated by PCR and cloned into pQE-30 and pCold I expression vectors, and then the recombinant flagellins of two strains were overexpressed in E. coli JM109 and pG-Tf/BL21, respectively. The molecular weight of the purified recombinant flagellins of NUF251 and NUF194 strains were estimated to be 45 kDa and 37 kDa, respectively on SDS-PAGE analysis. Referring the three-dimensional structure of Salmonella flagellin, which has been reported to have 4 domains (D0, D1, D2, and D3), high sequence homology between two flagellins of E. tarda was observed at conservative domain (D0 and D1) regions, whereas the sequences equivalent to D2 and D3 domains were different, and even equivalent to 57 amino acids were deleted in NUF194. Both recombinant flagellins induced NO production, mRNA expression level of inducible NO synthase (iNOS), and intercellular ROS production in mouse macrophage cell line RAW264.7 cells. Also, the secretion of TNF-α and its mRNA expression level were increased by treatment of both recombinant flagellins. These results indicate that the recombinant flagellins from different virulent E. tarda strains can stimulate macrophages with nearly equal levels as judged by the parameters tested, even though they are differences in the structure and molecular weight, suggesting that conservative D0 and D1 domains are sufficient structural elements for the recombinant flagellins to induce a certain level of macrophage-stimulation in vitro. Further studies are necessary focusing on the role of D2 and D3 domain regions of the recombinant flagellins as macrophage-stimulating agent as well as their influence on host immune system in vivo.

A prospective evaluation of postoperative swallowing function and dysphagia following endoscopic laryngopharyngeal surgery.

BACKGROUND: Endoscopic laryngopharyngeal surgery (ELPS) is a minimally invasive transoral surgery for superficial pharyngeal and laryngeal cancer, but dysphagia occasionally occurs post-treatment. We investigated dysphagia following ELPS and its risk factors. METHODS: Of the 145 patients who underwent ELPS, 92 were evaluated in this study using the Hyodo score, Functional Outcome Swallowing Scale, Eating Assessment Tool-10 along with the total scores for the three items of the method of intake, time, and food preoperatively and on postoperative 1, 3, and 6 months. We examined the 6-month trends of these values. Furthermore, the fasting period post-surgery, the need for swallowing rehabilitation by a speech therapist, and postoperative pneumonia episodes were set as outcomes reflecting the short-term swallowing function. We determined the associations between these outcomes and patient background factors. RESULTS: Postoperatively, the Hyodo score worsened at 1 month but recovered at 3 months. The Hyodo scores of all patients who underwent postcricoid ELPS did not worsen. The diameter of the resected specimen (DRS) was significantly associated with the need for swallowing rehabilitation and postoperative fasting time. A DRS ≥ 35 mm was considered the threshold for the need of swallowing rehabilitation, postoperative pneumonia, and prolonged postoperative fasting time. CONCLUSION: ELPS exerts a temporal and limited impact on the swallowing function, which recovers within 3 months in every swallowing evaluation. This necessitates additional care during the treatment of patients with mucosal defects ≥ 35 mm, owing to the significant association between the DRS and short-term swallowing function.

Gestational age-related changes in cervical gland length in normal singleton pregnancies from 17 to 36 weeks of gestation.

AIM: To determine the gestational age-related changes in cervical gland length in relation to cervical length (CL) in normal singleton pregnancies. METHODS: We studied 363 women with an uncomplicated singleton pregnancy (188 nulliparous and 175 multiparous women with one or more previous transvaginal deliveries). A total of 1138 cervical gland and CLs were measured longitudinally at 17-36 weeks of gestation using transvaginal ultrasonography along the curvature from the external os to the lower uterine segment and the internal end of the cervical gland area (CGA), respectively. Gestational age-related changes in cervical gland and CLs and their relationships were analyzed using a linear mixed model. RESULTS: Cervical gland and CLs decreased in different ways with advancing gestation depending on parity, and their changes were related to each other. The CGAs in nulliparous women were longer than those in multiparous women at 17-25 weeks of gestation (p < 0.05), but with no differences thereafter. CLs in multiparous women were different from those in nulliparous women at 17-23 and 35-36 weeks (p < 0.05), but there were no differences at 24-34 weeks. The cervix did not shorten compared with the CGA throughout the observational periods in nulliparous and multiparous women. CONCLUSIONS: Shortening of the cervix indicates changes to the lower uterine segment in normal pregnancies. The cervical gland region can be a useful marker representing the true cervix beyond 25 weeks of gestation, irrespective of parity.

Molecular Mechanisms of Neurogenic Lower Urinary Tract Dysfunction after Spinal Cord Injury.

This article provides a synopsis of current progress made in fundamental studies of lower urinary tract dysfunction (LUTD) after spinal cord injury (SCI) above the sacral level. Animal models of SCI allowed us to examine the effects of SCI on the micturition control and the underlying neurophysiological processes of SCI-induced LUTD. Urine storage and elimination are the two primary functions of the LUT, which are governed by complicated regulatory mechanisms in the central and peripheral nervous systems. These neural systems control the action of two functional units in the LUT: the urinary bladder and an outlet consisting of the bladder neck, urethral sphincters, and pelvic-floor striated muscles. During the storage phase, the outlet is closed, and the bladder is inactive to maintain a low intravenous pressure and continence. In contrast, during the voiding phase, the outlet relaxes, and the bladder contracts to facilitate adequate urine flow and bladder emptying. SCI disrupts the normal reflex circuits that regulate co-ordinated bladder and urethral sphincter function, leading to involuntary and inefficient voiding. Following SCI, a spinal micturition reflex pathway develops to induce an overactive bladder condition following the initial areflexic phase. In addition, without proper bladder-urethral-sphincter coordination after SCI, the bladder is not emptied as effectively as in the normal condition. Previous studies using animal models of SCI have shown that hyperexcitability of C-fiber bladder afferent pathways is a fundamental pathophysiological mechanism, inducing neurogenic LUTD, especially detrusor overactivity during the storage phase. SCI also induces neurogenic LUTD during the voiding phase, known as detrusor sphincter dyssynergia, likely due to hyperexcitability of Aδ-fiber bladder afferent pathways rather than C-fiber afferents. The molecular mechanisms underlying SCI-induced LUTD are multifactorial; previous studies have identified significant changes in the expression of various molecules in the peripheral organs and afferent nerves projecting to the spinal cord, including growth factors, ion channels, receptors and neurotransmitters. These findings in animal models of SCI and neurogenic LUTD should increase our understanding of pathophysiological mechanisms of LUTD after SCI for the future development of novel therapies for SCI patients with LUTD.

High-risk patent foramen ovale and elderly in cryptogenic stroke.

BACKGROUND: High-risk patent foramen ovale (PFO) could be pathological in cryptogenic stroke (CS), but its clinical characteristics have not been fully studied, especially in elderly patients. METHODS: Patients with CS were enrolled in the CHALLENGE ESUS/CS registry, a multicenter registry of CS patients undergoing transesophageal echocardiography. Clinical characteristics were compared among three groups: high-risk PFO group, large shunt PFO (≥25 microbubbles) or PFO with atrial septal aneurysm (ASA); right-to-left shunt (RLS) group, RLS including PFO with <25 microbubbles or without ASA; and no-RLS group. RESULTS: In total, 654 patients were analyzed: 91, 221, and 342 in the high-risk PFO, RLS, and no-RLS groups, respectively. In multinomial logistic regression analysis, the male sex (odds ratio [OR] 1.825 [1.067-3.122]) was independently associated with high-risk PFO, but hypertension (OR, 0.562 [0.327-0.967]), multiple infarctions (OR, 0.601 [0.435-0.830]), and other cardioaortic embologenic risks (OR, 0.514 [0.294-0.897]) were inversely associated with high-risk PFO compared with non-RLS. In 517 patients aged ≥60 years, multiple infarctions (OR, 0.549 [0.382-0.788]) and other cardioaortic embologenic risks (OR, 0.523 [0.286-0.959]) were inversely associated with high-risk PFO. CONCLUSIONS: High-risk PFO had specific clinical characteristics and possible mechanistic associations, and this trend was consistent among CS patients aged ≥60 years. CLINICAL TRIAL REGISTRATION INFORMATION: http://www.umin.ac.jp/ctr/ (UMIN000032957).

Systematic Classification of Curvature and Feature Descriptor of 3D Shape and Its Application to "Complexity" Quantification Methods.

Generative design is a system that automates part of the design process, but it cannot evaluate psychological issues related to shapes, such as "beauty" and "liking". Designers therefore evaluate and choose the generated shapes based on their experience. Among the design features, "complexity" is considered to influence "aesthetic preference". Although feature descriptors calculated from curvature can be used to quantify "complexity", the selection guidelines for curvature and feature descriptors have not been adequately discussed. Therefore, this study aimed to conduct a systematic classification of curvature and a feature descriptor of 3D shapes and to apply the results to the "complexity" quantification. First, we surveyed the literature on curvature and feature descriptors and conducted a systematic classification. To quantify "complexity", we used five curvatures (Gaussian curvature, mean curvature, Casorati curvature, shape index, and curvature index) and a feature descriptor (entropy of occurrence probability) obtained from the classification and compared them with the sensory evaluation values of "complexity". The results showed that the determination coefficient between the quantified and sensory evaluation values of "complexity" was highest when the mean curvature was used. In addition, the Casorati curvature tended to show the highest signal-to-noise ratio (i.e., a high determination coefficient irrespective of the parameters set in the entropy calculation). These results will foster the development of generative design of 3D shapes using psychological evaluation.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

Stimulation of brain corticotropin-releasing factor receptor type1 facilitates the rat micturition via brain glutamatergic receptors.

Corticotropin-releasing factor (CRF), a representative stress-related neuropeptide, in the central nervous system reportedly both facilitates and suppresses the micturition, therefore, roles of central CRF in regulation of the micturition are still controversial. In this study, we investigated (1) effects of intracerebroventricularly (icv)-administered CRF on the micturition, and (2) brain CRF receptor subtypes (CRFR1/CRFR2) and glutamatergic receptors (NMDA/AMPA subtypes) involved in the CRF-induced effects in male Wistar rats under urethane anesthesia. Intercontraction intervals (ICI), and maximal voiding pressure (MVP), were evaluated by continuous cystometry 45 min before CRF administration or intracerebroventricular pretreatment with other drugs as follows and 3 h after CRF administration. Single-voided volume (Vv), post-voiding residual volume (Rv), bladder capacity (BC), and voiding efficiency (VE) were evaluated by single cystometry 60 min before CRF administration and 60-120 min after the administration. Icv-administered CRF reduced ICI, Vv, and BC without changing MVP, Rv, or VE. The CRF-induced ICI reduction was attenuated by icv-pretreated CP154526 (CRFR1 antagonist), MK-801 (NMDA receptor antagonist), and DNQX (AMPA receptor antagonist), but not by K41498 (CRFR2 antagonist). These results indicate that stimulation of brain CRFR1 can be involved in facilitation of the rat micturition via brain NMDA/AMPA receptors.

Stepwise generation of AID knock-in and conditional knockout mice from a single gene-targeting event.

Activation-induced cytidine deaminase (AID) encoded by the Aicda gene initiates class-switch recombination and somatic hypermutation of immunoglobulin genes. In addition to this function, AID is also implicated in the epigenetic regulation in pluripotent stem cells and in the oncogenesis of lymphoid and non-lymphoid origins. To examine AID's role in specific cell types, we developed mouse strains of conditional knockout (Aicda-FL) and knock-in with a red fluorescent protein gene (RFP) inserted into the Aicda locus (Aicda-RFP). These two strains were obtained from a single targeting event in embryonic stem cells by a three-loxP or tri-lox strategy. Partial and complete recombination among the three loxP sites in the Aicda-RFP locus gave rise to Aicda-FL and AID-deficient loci (Aicda-KO), respectively, after mating Aicda-RFP mice with Cre-expressing mice driven by tissue-non-specific alkaline phosphate promoter. We confirmed RFP expression in B cells of germinal centers of intestine-associated lymphoid tissue. Mice homozygous for each allele were obtained and were checked for AID activity by class-switch and hypermutation assays. AID activity was normal for Aicda-FL but partially and completely absent for Aicda-RFP and Aicda-KO, respectively. Aicda-FL and Aicda-RFP mice would be useful for studying AID function in subpopulations of B cells and in non-lymphoid cells.

Quantitative Evaluation of Cerebellar Function in Multiple System Atrophy with Transcranial Magnetic Stimulation.

Objective evaluation of cerebellar dysfunction in neurodegenerative disorders is often difficult because of other overlapping symptoms. Cerebellar inhibition (CBI) tested by dual-coil transcranial magnetic stimulation (TMS) is anticipated as a promising measure to estimate cerebellar function. Cerebellar TMS inhibits the primary motor cortex (M1), which can be measured as the decrease of motor evoked potential (MEP) elicited by a single-pulse TMS over the M1. This study was conducted to quantify cerebellar dysfunction using CBI in cerebellar type multiple system atrophy (MSA-C) patients. First, CBI was measured using MEP elicited from a hand muscle by stimulating the hand motor area of M1. The amount of CBI was defined as the degree of decrease in the MEP amplitude in the presence of cerebellar stimulation compared with the condition of M1 stimulation alone. Results of the MSA-C patients were compared with those of healthy volunteers. Correlation between amounts of CBI and a clinical scale of ataxia, the International Cooperative Ataxia Scale Rating (ICARS), was assessed. Healthy volunteers showed more inhibition than MSA-C patients. Moreover, ICARS showed that the CBI amount in the patients is correlated with the degree of ataxia significantly. Results suggest that CBI can be a good marker of disease progression in MSA-C patients.

Protective effects of hydrogen sulfide pretreatment on cyclophosphamide-induced bladder dysfunction in rats via suppression of bladder afferent nerves.

Cyclophosphamide (CYP), a broad-spectrum anticancer drug, causes serious side effects, such as haemorrhagic cystitis (HC). Hydrogen sulfide (H2S), an endogenous gasotransmitter, has physiological properties, including anti-inflammation, anti-oxidation, and neuromodulation. In this study, we investigated the effects of NaHS (H2S donor) pretreatment on bladder dysfunction in CYP-treated rats. Male Wistar rats were intraperitoneally pretreated with NaHS (3 or 10 µmol/kg) or vehicle once daily for 7 days before cystometry, and CYP (150 mg/kg) or saline was intraperitoneally administered 2 days before cystometry. After cystometry, the bladder tissues were collected for haematoxylin and eosin staining. In some rats, capsaicin (CAP), which can desensitise CAP-sensitive afferent nerves, was subcutaneously injected at 125 mg/kg 4 days before cystometry. CYP reduced intercontraction intervals (ICI) and bladder compliance (Comp) and increased the number of non-voiding contractions (NVCs) compared with the saline-treated control group. NaHS pretreatment dose-dependently improved the CYP-induced these changes. In bladder tissues, CYP increased histological scores of neutrophil infiltration, haemorrhage, and oedema, while NaHS had no effect on these CYP-induced changes. CAP showed a tendency to suppress CYP-induced changes in ICI. NaHS-induced improvement in CYP-induced changes in urodynamic parameters were not detected in CAP-treated rats. These findings suggest that NaHS pretreatment prevented bladder dysfunction in CYP-treated rats by suppressing CAP-sensitive bladder afferent nerves, but not by suppressing bladder inflammation. Therefore, H2S represents a new candidate as a protective drug for bladder dysfunction induced by HC, a side effect of CYP chemotherapy.