Michiko Nakamura

Signaling complex formation of phospholipase Cβ4 with metabotropic glutamate receptor type 1α and 1,4,5-trisphosphate receptor at the perisynapse and endoplasmic reticulum in the mouse brain

Upon activation of cell surface receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC) β hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5‐trisphosphate (IP3) and 1,2‐diacylglycerol. PLCβ4 has been characterized as the isoform enriched in cerebellar Purkinje cells (PCs) and the retina and involved in motor and visual functions. Here we examined cellular and subcellular distributions of PLCβ4 in adult mouse brains. Immunohistochemistry showed that high levels of PLCβ4 were detected in the somatodendritic domain of neuronal populations expressing the metabotropic glutamate receptor (mGluR) type 1α, including olfactory periglomerular cells, neurons in the bed nucleus anterior commissure, thalamus, substantia nigra, inferior olive, and unipolar brush cells and PCs in the cerebellum. Low to moderate levels were detected in many other mGluR1α‐positive neurons and in a few mGluR1α‐negative neurons. In PCs, immunogold electron microscopy localized PLCβ4 to the perisynapse, at which mGluR1α is concentrated, and to the smooth endoplasmic reticulum in dendrites and spines, an intracellular Ca2+ store gated by IP3 receptors. In the cerebellum, immunoblot demonstrated its concentrated distribution in the post‐synaptic density and microsomal fractions, where mGluR1α and type 1 IP3 receptor were also greatly enriched. Furthermore, PLCβ4 formed coimmunoprecipitable complexes with mGluR1α, type 1 IP3 receptor and Homer 1. These results suggest that PLCβ4 is preferentially localized in the perisynapse and smooth endoplasmic reticulum as a component of the physically linked phosphoinositide signaling complex. This close molecular relationship might provide PLCβ4 with a high‐fidelity effector function to mediate various neuronal responses under physiological and pathophysiological conditions.

Patterns of expression for the mRNA corresponding to the four isoforms of phospholipase Cbeta in mouse brain

Ligand binding to neurotransmitter and hormone receptors which couple to the Gq subclass of GTP‐binding protein leads to the activation of phospholipase Cβ (PLCβ) which hydrolyses phosphatidyl‐inositol 4,5‐bisphosphate, yielding a pair of second messengers, diacylglycerol and inositol 1,4,5‐trisphosphate (IP3). The expression of PLCβ1–4 mRNAs was comparatively examined by in situ hybridization in the mouse brain. In adults, PLCβ1 mRNA was expressed predominantly in the telencephalon, including the cerebral cortex, hippocampus, amygdala, lateral septum and olfactory bulb, with little expression in most thalamic nuclei. PLCβ2 mRNA was distributed in the white matter, suggesting its expression in non‐neuronal cells, most likely oligodendrocytes. PLCβ3 mRNA was specifically expressed in cerebellar Purkinje cells. The highest levels of PLCβ4 mRNA were detected in Purkinje cells. High levels of PLCβ4 mRNA were also found in the thalamus and medial septum, whereas weak signals were detected in most telencephalic regions, thus showing an expression pattern almost reciprocal to that of PLCβ1 mRNA. During development, such characteristic regional expression of PLCβ1 and PLCβ4 were observed starting in late foetal stages, while specific expression of PLCβ2 and PLCβ3 appeared in early postnatal stages. We conclude that despite the existence of four PLCβ isoforms, only one or two of them is expressed in individual neurons and glial cells. The distinct expression of PLCβs provides a molecular basis for analysing the nature of the specific signal transduction pathway leading to the production of diacylglycerol and IP3 in distinct cell types and in different regions of the brain.

Mamld1 Knockdown Reduces Testosterone Production and Cyp17a1 Expression in Mouse Leydig Tumor Cells

Background MAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments. Methodology/Principal Findings Mamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs. Conclusions/Significance The results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling.

Mamld1 Deficiency Significantly Reduces mRNA Expression Levels of Multiple Genes Expressed in Mouse Fetal Leydig Cells but Permits Normal Genital and Reproductive Development

Although mastermind-like domain containing 1 (MAMLD1) (CXORF6) on human chromosome Xq28 has been shown to be a causative gene for 46,XY disorders of sex development with hypospadias, the biological function of MAMLD1/Mamld1 remains to be elucidated. In this study, we first showed gradual and steady increase of testicular Mamld1 mRNA expression levels in wild-type male mice from 12.5 to 18.5 d postcoitum. We then generated Mamld1 knockout (KO) male mice and revealed mildly but significantly reduced testicular mRNA levels (65-80%) of genes exclusively expressed in Leydig cells (Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3) as well as grossly normal testicular mRNA levels of genes expressed in other cell types or in Leydig and other cell types. However, no demonstrable abnormality was identified for cytochrome P450 17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B) protein expression levels, appearance of external and internal genitalia, anogenital distance, testis weight, Leydig cell number, intratesticular testosterone and other steroid metabolite concentrations, histological findings, in situ hybridization findings for sonic hedgehog (the key molecule for genital tubercle development), and immunohistochemical findings for anti-Müllerian hormone (Sertoli cell marker), HSD3B (Leydig cell marker), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (germ cell marker) in the KO male mice. Fertility was also normal. These findings imply that Mamld1 deficiency significantly reduces mRNA expression levels of multiple genes expressed in mouse fetal Leydig cells but permits normal genital and reproductive development. The contrastive phenotypic findings between Mamld1 KO male mice and MAMLD1 mutation positive patients would primarily be ascribed to species difference in the fetal sex development.

Long-Term Outcome of Pituitary-Gonadal Axis and Gonadal Growth in Patients With Hypospadias at Puberty

PURPOSE Reports of pubertal hormonal and gonadal status in patients with hypospadias are scarce. We evaluated the pituitary-gonadal axis and gonadal growth in patients with hypospadias at puberty. MATERIALS AND METHODS We retrospectively reviewed serum luteinizing hormone, follicle-stimulating hormone, testosterone and testicular volume at puberty (age 15 years or greater) in the medical charts of patients with hypospadias treated since 1986 and followed at our institution. RESULTS Enrolled in this study were 43 patients with a mean age at evaluation of 17.6 years (range 15.1 to 22.8). Of these patients 14 and 29 were treated for mild and severe hypospadias, respectively. Six patients with severe hypospadias underwent bilateral orchiopexy for bilateral undescended testes. All patients were Tanner stage 4 to 5 at evaluation. Of 14 mild hypospadias cases we noted hypergonadotropic hypogonadism, hypogonadotropic hypogonadism, decreased luteinizing hormone and decreased testosterone in 1 each (7% each). Of 23 severe hypospadias cases without bilateral undescended testes we noted hypergonadotropic hypogonadism, partial androgen insensitivity syndrome and decreased testosterone in 2 (9%), 1 (4%) and 1 (4%), respectively. Of 6 patients with severe hypospadias and bilateral undescended testes we noted hypergonadotropic hypogonadism in 1 (17%) and increased luteinizing hormone with normal testosterone in 2 (33%). Testicular volume less than 10 ml with increased follicle-stimulating hormone was identified in 7 of 43 patients, including 1 of 14 (7%) with mild hypospadias, 3 of 23 (13%) with severe hypospadias without bilateral undescended testes and 3 of 6 (50%) with severe hypospadias and bilateral undescended testes. CONCLUSIONS Our study revealed endocrine dysfunction in patients with mild and severe hypospadias at puberty even without an undescended testis. Of these patients those with severe hypospadias and an undescended testis may be at higher risk for impaired spermatogenesis.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

Early Discontinuation of Antibiotic Prophylaxis in Patients with Persistent Primary Vesicoureteral Reflux Initially Detected during Infancy: Outcome Analysis and Risk Factors for Febrile Urinary Tract Infection

PURPOSE We retrospectively assessed the incidence of and risk factors for febrile urinary tract infection in children during active surveillance after early discontinuation of antibiotic prophylaxis. MATERIALS AND METHODS We retrospectively evaluated 9 females and 61 uncircumcised males diagnosed with primary vesicoureteral reflux before age 1 year who had persistent reflux on followup voiding cystourethrogram and were subsequently followed under active surveillance without continuous antibiotic prophylaxis. Patients with secondary vesicoureteral reflux or associated urological abnormality were excluded. Clinical outcomes, including incidence of febrile urinary tract infection and new scar formation, were evaluated. Risk factors for febrile urinary tract infection were also analyzed. RESULTS Mean age at stopping continuous antibiotic prophylaxis was 21 months, and mean followup was 61 months. During active surveillance 21 patients had febrile urinary tract infection, and the 5-year infection-free rate under active surveillance was 67.5%. One or 2 foci of minimal new scarring developed in 4 of 16 patients who underwent followup dimercapto-succinic acid scan after febrile urinary tract infection. On multivariate analysis dilated vesicoureteral reflux on followup voiding cystourethrogram was the only significant risk factor for febrile urinary tract infection. CONCLUSIONS This study revealed that about two-thirds of patients with persistent vesicoureteral reflux were free of febrile urinary tract infection during 5 years of active surveillance. Those with dilated vesicoureteral reflux on followup voiding cystourethrogram are at significantly greater risk for febrile urinary tract infection. Accordingly active surveillance, especially in patients with nondilated vesicoureteral reflux on followup voiding cystourethrogram, seems to be a safe option even in children who have not yet been toilet trained.

Novel Splice Site Mutation in MAMLD1 in a Patient with Hypospadias.

MAMLD1 is a causative gene for disorders of sex development. Several MAMLD1 mutations have been shown to cause hypospadias by generating dysfunctional proteins and/or unstable mRNAs. Here, we identified an intronic mutation of MAMLD1 (g.IVS4−2A>G) in 1 of 180 hypospadias patients. RT-PCR of the patients skin sample showed normal expression of full-length MAMLD1 and markedly reduced expression of a known splice variant lacking exon 4. A hitherto unreported splice variant that lacks exon 5 was similarly identified in samples of the patient and control individuals. The full-length transcript of the patient contained mutant mRNA lacking the first 10 nucleotides of exon 5 (c.1822_1831delACTCATGTAG, p.K609fsX1070). In vitro assays using cells expressing the full-length wild-type and mutant proteins revealed reduced expression of the mutant. The expression of the wild-type and mutant MAMLD1 showed parallel changes upon treatment with a proteasome inhibitor and a translation inhibitor. The mutant-expressing cells exerted low transactivation activity for the Hes3 promoter, which reflected limited expression of the mutant protein. These results imply that the pathogenic events resulting from MAMLD1 mutations include splice errors. Furthermore, this study raises the possibility of translation failure of MAMLD1 mutants, which deserves further investigation.

Abnormal dimercapto-succinic acid scan is a predictive factor of breakthrough urinary tract infection in children with primary vesicoureteral reflux.

PURPOSE We investigated factors affecting the breakthrough urinary tract infection rate during prophylactic antibiotic treatment in children with primary vesicoureteral reflux. MATERIALS AND METHODS Medical charts were retrospectively reviewed in children with primary vesicoureteral reflux diagnosed at age 12 months or less who received prophylactic antibiotics and underwent (99m)Tc-dimercapto-succinic acid scan. Parameters assessed for their relation to breakthrough urinary tract infection were gender, presenting symptoms, age at presentation, prophylactic antibiotic type, reflux grade at presentation and scan findings. RESULTS Enrolled in the study were 52 boys and 6 girls with a mean age at presentation of 3.7 months and a mean followup of 42.5 months. Urinary tract infection was a presenting symptom in 46 children. Low reflux grade (1-3) was identified in 18 children and 40 had high grade reflux (4-5). Abnormal (99m)Tc-dimercapto-succinic acid scan was documented in 36 children (62%). During followup breakthrough vesicoureteral reflux developed in 12 children, including 11 of 36 (31%) with an abnormal scan but only 1 of 22 (5%) with a normal scan (p = 0.021). The breakthrough urinary tract infection-free rate during followup was significantly lower in children with an abnormal scan (p = 0.033). Other factors were not significantly associated with the breakthrough urinary tract infection rate. CONCLUSIONS Abnormal (99m)Tc-dimercapto-succinic acid scan may be a factor predicting breakthrough urinary tract infection in children with primary vesicoureteral reflux. Prophylactic antibiotics may have a limited treatment role in children with an abnormal scan.

Long-Term Outcome of Vaginoplasty With the Bilateral Labioscrotal Flap

PURPOSE We report the long-term outcome of vaginoplasty with the bilateral labioscrotal flap with special emphasis on vaginal stenosis. MATERIALS AND METHODS Of 23 children with ambiguous genitalia and low vaginal entry who underwent vaginoplasty between January 1985 and July 2003 with the bilateral labioscrotal flap 13 followed more than 5 years after surgery who were 10 years old or older at the most recent evaluation were included in this long-term outcome study. Vaginal caliber was estimated according to a previously described assessment system adopted for vaginoplasty results. RESULTS The underlying disease was congenital adrenal hyperplasia in 11 cases, mixed gonadal dysgenesis in 1 and ovotesticular sexual development disorder in 1. Mean age at vaginoplasty and at the most recent evaluation was 3.8 (range 2.0 to 12.9) and 14.6 years (range 10.9 to 21.5), respectively. Vaginal caliber at the most recent evaluation was adequate in 6 patients (46%), stenotic in 5 (39%) and strictured in 2 (15%). Three of the 7 patients diagnosed with stricture or stenosis were diagnosed at age less than 12 years. One of these patients diagnosed with stricture was treated with dilation and the other 2 patients were observed. These patients had no trouble with menstruation. Four patients diagnosed with stricture or stenosis at age 14 years or older were treated surgically with dilation in 1 and perineal flap vaginoplasty in 3. They showed adequate vaginal caliber at 3 to 31 months of followup. In 7 patients evaluated at the beginning of puberty and several years later vaginal caliber had enlarged in 5 but remained unchanged in 2. CONCLUSIONS To our knowledge this is the first report of the long-term outcome of vaginoplasty with the bilateral labioscrotal flap. Although vaginal stenosis/stricture was observed at puberty in about half of the patients, severe stricture was uncommon. Serial evaluation for vaginal stenosis/stricture at the beginning of puberty for menstruation and several years later for vaginal intercourse is recommended in patients treated with vaginal reconstruction.