A method for calculating left ventricular ejection fraction noninvasively from left ventricular arterial coupling (Ees/Ea).

BACKGROUND: Ejection fraction (EF), which is assessed using ultrasonography, is a standard parameter for evaluating cardiac function in clinical cardiology and for cardiovascular management during general anesthesia. However, it is impossible to continuously and non-invasively assess EF using ultrasonography. The aim of our study was to develop a method for estimating EF non-invasively using the left ventricular arterial coupling ratio (Ees/Ea). METHODS: Ees/Ea was estimated non-invasively using the parameters pre-ejection period (PEP), ejection time (ET), end-systolic pressure (Pes) and diastolic pressure (Pad), which were calculated from a vascular screening system, VeSera 1000/1500 (Fukuda Denshi Co., Ltd., Tokyo, Japan). Then, left ventricular efficiency (Eff) as a pump, defined as the ratio of external work (EW) to myocardial oxygen consumption, which strongly correlates with the pressure-volume area (PVA), was calculated by a new formula using Ees/Ea, and was used to approximate EF (EFeff). Simultaneously, we measured EF using transthoracic echocardiography (EFecho), and compared it with EFeff. RESULTS: The study included 44 healthy adults (36 males, 8 females), in whom mean EFecho was 66 ± 5% and EFeff was 57 ± 9%. We found a positive correlation between EFecho and EFeff (R2 = 0.219, p < 0.05) on Bland-Altman analysis, with limits of agreement of - 7.5 to 24.4%, and percentage error of 24%. CONCLUSION: The results suggest that EF can be measured non-invasively using left ventricular arterial coupling.

Deletion of Orc4 during oogenesis severely reduces polar body extrusion and blocks zygotic DNA replication†.

Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.

The association between initial calculated driving pressure at the induction of general anesthesia and composite postoperative oxygen support.

PURPOSE: Early discontinuation of postoperative oxygen support (POS) would partially depend on the innate pulmonary physics. We aimed to examine if the initial driving pressure (dP) at the induction of general anesthesia (GA) predicted POS prolongation. METHODS: We conducted a single-center retrospective study using the facility's database. Consecutive subjects over 2 years were studied to determine the change in odds ratio (OR) for POS prolongation of different dP classes at GA induction. The dP (cmH2O) was calculated as the ratio of tidal volume (mL) over dynamic Crs (mL/cmH2O) regardless of the respiratory mode. The adjusted OR was calculated using the logistic regression model of multivariate analysis. Moreover, we performed a secondary subgroup analysis of age and the duration of GA. RESULTS: We included 5,607 miscellaneous subjects. Old age, high scores of American Society of Anesthesiologist physical status, initial dP, and long GA duration were associated with prolonged POS. The dP at the induction of GA (7.78 [6.48, 9.45] in median [interquartile range]) was categorized into five classes. With the dP group of 6.5-8.3 cmH2O as the reference, high dPs of 10.3-13 cmH2O and ≥ 13 cmH2O were associated with significant prolongation of POS (adjusted OR, 1.62 [1.19, 2.20], p = 0.002 and 1.92 [1.20, 3.05], p = 0.006, respectively). The subgroup analysis revealed that the OR for prolonged POS of high dPs disappeared in the aged and ≥ 6 h anesthesia time subgroup. CONCLUSIONS: High initial dPs ≥ 10 cmH2O at GA induction predicted longer POS than those of approximately 7 cmH2O. High initial dPs were, however, a secondary factor for prolongation of postoperative hypoxemia in old age and prolonged surgery.

Selenoprotein I is essential for murine embryogenesis.

Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI-/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.

Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors.

Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/).

Dose-dependent functions of fibroblast growth factor 9 regulate the fate of murine XY primordial germ cells.

Male differentiation of primordial germ cells (PGCs) is initiated by the inhibition of entry into meiosis and exposure to male-inducing factor(s), which are regulated by somatic elements of the developing gonad. Fibroblast growth factor 9 (FGF9) produced by pre-Sertoli cells is essential for male gonadal differentiation and also contributes to survival and male differentiation of XY PGCs. However, it is not clear how FGF9 regulates PGC fate. Using a PGC culture system, we identified dose-dependent, fate-determining functions of FGF9 in XY PGCs. Treatment with low levels of FGF9 (0.2 ng/ml) increased expression of male-specific Dnmt3L and Nanos2 in XY PGCs. Conversely, treatment with high levels of FGF9 (25 ng/ml) suppressed male-specific gene expression and stimulated proliferation of XY PGCs. Western blotting showed that low FGF9 treatment enhanced p38 MAPK (mitogen-activated protein kinase) phosphorylation in the same cells. In contrast, high FGF9 treatment significantly stimulated the ERK (extracellular signal-regulated kinase)1/2 signaling pathway in XY PGCs. We investigated the relationship between the ERK1/2 signaling pathway stimulated by high FGF9 and regulation of PGC proliferation. An ERK1/2 inhibitor (U0126) suppressed the PGC proliferation that would otherwise be stimulated by high FGF9 treatment, and increased Nanos2 expression in XY PGCs. Conversely, a p38 MAPK inhibitor (SB202190) significantly suppressed Nanos2 expression that would otherwise be stimulated by low FGF9 in XY PGCs. Taken together, our results suggest that stage-specific expression of FGF9 in XY gonads regulates the balance between proliferation and differentiation of XY PGCs in a dose-dependent manner.

TOMATOMA Update: Phenotypic and Metabolite Information in the Micro-Tom Mutant Resource.

TOMATOMA (http://tomatoma.nbrp.jp/) is a tomato mutant database providing visible phenotypic data of tomato mutant lines generated by ethylmethane sulfonate (EMS) treatment or γ-ray irradiation in the genetic background of Micro-Tom, a small and rapidly growing variety. To increase mutation efficiency further, mutagenized M3 seeds were subjected to a second round of EMS treatment; M3M1 populations were generated. These plants were self-pollinated, and 4,952 lines of M3M2 mutagenized seeds were generated. We checked for visible phenotypes in the M3M2 plants, and 618 mutant lines with 1,194 phenotypic categories were identified. In addition to the phenotypic information, we investigated Brix values and carotenoid contents in the fruits of individual mutants. Of 466 samples from 171 mutant lines, Brix values and carotenoid contents were between 3.2% and 11.6% and 6.9 and 37.3 µg g(-1) FW, respectively. This metabolite information concerning the mutant fruits would be useful in breeding programs as well as for the elucidation of metabolic regulation. Researchers are able to browse and search this phenotypic and metabolite information and order seeds of individual mutants via TOMATOMA. Our new Micro-Tom double-mutagenized populations and the metabolic information could provide a valuable genetic toolkit to accelerate tomato research and potential breeding programs.

NIG_MoG: a mouse genome navigator for exploring intersubspecific genetic polymorphisms.

The National Institute of Genetics Mouse Genome database (NIG_MoG; http://molossinus.lab.nig.ac.jp/msmdb/) primarily comprises the whole-genome sequence data of two inbred mouse strains, MSM/Ms and JF1/Ms. These strains were established at NIG and originated from the Japanese subspecies Mus musculus molossinus. NIG_MoG provides visualized genome polymorphism information, browsing single-nucleotide polymorphisms and short insertions and deletions in the genomes of MSM/Ms and JF1/Ms with respect to C57BL/6J (whose genome is predominantly derived from the West European subspecies M. m. domesticus). This allows users, especially wet-lab biologists, to intuitively recognize intersubspecific genome divergence in these mouse strains using visual data. The database also supports the in silico screening of bacterial artificial chromosome (BAC) clones that contain genomic DNA from MSM/Ms and the standard classical laboratory strain C57BL/6N. NIG_MoG is thus a valuable navigator for exploring mouse genome polymorphisms and BAC clones that are useful for studies of gene function and regulation based on intersubspecific genome divergence.

Primary epimutations introduced during intracytoplasmic sperm injection (ICSI) are corrected by germline-specific epigenetic reprogramming.

The use of assisted reproductive technologies (ART) has become increasingly common worldwide and is now responsible for 2-3% of children born in developed countries. Multiple reports have suggested that ART-conceived children are more likely to develop rare epigenetic disorders such as Beckwith-Wiedemann Syndrome or Angelman Syndrome, both of which involve dysregulation of imprinted genes. Anecdotal reports suggest that animals produced with ART that manifest apparent epigenetic defects typically do not transmit these epimutations to subsequent generations when allowed to breed naturally, but this hypothesis has not been directly studied. We analyzed allele-specific DNA methylation and expression at three imprinted genes, H19, Snrpn, and Peg3, in somatic cells from adult mice generated with the use of intracytoplasmic sperm injection (ICSI), a type of ART. Epimutations were detected in most of the ICSI-derived mice, but not in somatic cells of their offspring produced by natural mating. We examined germ cells from the ICSI mice that exhibited epimutations in their somatic cells and confirmed normal epigenetic reprogramming of the three imprinted genes analyzed. Collectively, these results confirm that ART procedures can lead to the formation of primary epimutations, but while such epimutations are likely to be maintained indefinitely in somatic cells of the ART-derived individuals, they are normally corrected in the germ line by epigenetic reprogramming and thus, not propagated to subsequent generations.

Early-Morning Type Ventricular Fibrillation With J Wave.

A 67-year-old man who had cardiopulmonary arrest (CPA) at home was admitted to our institution. His spontaneous circulation was restored by bystander cardiopulmonary resuscitation (CPR) performed by his wife and an automated external defibrillator (AED). J waves were observed in the inferior leads of an electrocardiogram. We performed an implantable cardioverter defibrillator (ICD) implantation. After the ICD implantation, appropriate shocks due to ventricular fibrillation (VF) were observed on interrogation of the ICD at a frequency of twice a month. Most VF events occurred in the early morning between 1:00 to 6:00, and ventricular premature contractions (VPCs) were detected just before the occurrence of VF. Since the VF events always occurred in the early morning, we started long-acting disopyramide (150 mg/day, before bedtime), which has a muscarinic receptor blocking action. As a result, he has not received any appropriate ICD shocks for more than two years.

Efficacy of Dabigatran for Dissolving Deep Vein Thromboses in Outpatients With a Deteriorated General Condition.

Non-vitamin K antagonist oral anticoagulants (NOACs) have been widely used for the prevention of ischemic strokes in patients with nonvalvular atrial fibrillation (AF). At present, NOACs have been evaluated for the treatment of deep vein thrombosis (DVT). We examined the efficacy of dabigatran, the first NOAC for anticoagulation of AF in Japan, in outpatients who suffered from DVTs under a deteriorated general condition.Thirty-six consecutive outpatients diagnosed with DVT at our institute were enrolled. Not all patients could be hospitalized due to other clinical problems; 15 (42%) had malignant tumors, 9 (25%) psychological disorders, and 6 (17%) postoperative orthopedic disease. Dabigatran was administered at a dose of 110-150 mg once or twice daily, depending on the renal function and age. The mean dosage of dabigatran was 211.7 ± 36.6 mg per day. In 18 (50%) patients, the DVTs were completely dissolved and disappeared over a treatment term of 4.3 ± 4.3 months. In 9 patients (25%), the DVTs partly dissolved, but in the remaining 9 (25%) patients, dabigatran was totally ineffective. During a follow-up of 30.5 ± 5.3 months, DVTs did not recur with dabigatran in 18 patients with an effective efficacy. In a multivariate analysis, patients with small sized thromboses and those without malignant tumors were significantly associated with the DVTs dissolving (P = 0.003 and P = 0.006, respectively).Dabigatran was effective for dissolving DVTs in outpatients with a poor condition, particularly when the size of the DVT was small and malignant tumors were absent.

Gonadotropin stimulation contributes to an increased incidence of epimutations in ICSI-derived mice.

We previously demonstrated that intracytoplasmic sperm injection (ICSI), a type of assisted reproductive technology (ART), can induce epimutations and/or epimutant phenotypes in somatic tissues of adult mice produced by this method. In the present study, we compared the occurrence of epimutations in mice produced by natural conception, ICSI and somatic cell nuclear transfer. Surprisingly, we observed the highest frequency of epimutations in somatic tissues from ICSI-derived mice. We also observed a delay in reprogramming of the maternal allele of the imprinted H19 gene in spermatogonia from juvenile ICSI-derived male mice. These observations led us to hypothesize that the exposure of the maternal gametic genome to exogenous gonadotropins during the endocrine stimulation of folliculogenesis (superovulation) may contribute to the disruption of the normal epigenetic programming of imprinted loci in somatic tissues and/or epigenetic reprogramming in the germ line of ensuing offspring. To test this hypothesis, we uncoupled superovulation from ICSI by subjecting female mice to gonadotropin stimulation and then allowing them to produce offspring by natural mating. We found that mice produced in this way also exhibited epimutations and/or epimutant phenotypes in somatic tissues and delayed epigenetic reprogramming in spermatogenic cells, providing evidence that gonadotropin stimulation contributes to the induction of epimutations during ART procedures. Our results suggest that gonadotropin stimulation protocols used in conjunction with ART procedures should be optimized to minimize the occurrence of epimutations in offspring produced by these methods.

Comparison of Left Ventricular End-Diastolic Volume Approximated from Mean Blood Pressure and Stroke Volume and End-Diastolic Volume Calculated from Left Ventricular-Aortic Coupling.

Objectives: The purpose of this study was to compare left ventricular end-diastolic volume (EDV), derived from left ventricular arterial coupling (Ees/Ea), and mean arterial blood pressure. Both of these methods of measuring EDV require some invasive procedure. However, the method of measuring EDV approximate is less invasive than the EDV coupling measuring method. This is because EDV approximate only requires arterial pressure waveform as an invasive procedure. Methods: This study included 14 patients with normal cardiac function who underwent general anesthesia. The point when blood pressure stabilized after the induction of anesthesia was taken as a baseline according to the study protocol. At the point when systolic arterial blood pressure fell 10% or more from the baseline blood pressure, 300 mL of colloid solution was administered over 15 min. EDV approximate and EDV coupling were calculated for each of the 14 patients at three points during the course of anesthetic. Each value was obtained by calculating a 5 min average. The timing of these three points was 5 min before, 5 min during, and 5 min after infusion loading. Results: The total number of comparable points was 42; 3 points were taken from each of the 14 participants. Both EDV approximate and EDV coupling increased through the infusion load testing. Scatter plots were prepared, and regression lines were calculated from the obtained values. A high correlation was shown between EDV approximate and EDV coupling (R2 = 0.96, p < 0.05). Conclusions: In patients with good cardiac function, EDV approximate can be substituted for EDV coupling, suggesting the possibility that EDV can be continuously and less invasively calculated under the situation of general anesthesia.

Redox regulation of carbonic anhydrases via thioredoxin in chloroplast of the marine diatom Phaeodactylum tricornutum.

Thioredoxins (Trxs) are important regulators of photosynthetic fixation of CO(2) and nitrogen in plant chloroplasts. To date, they have been considered to play a minor role in controlling the Calvin cycle in marine diatoms, aquatic primary producers, although diatoms possess a set of plastidic Trxs. In this study we examined the influences of the redox state and the involvement of Trxs in the enzymatic activities of pyrenoidal carbonic anhydrases, PtCA1 and PtCA2, in the marine diatom Phaeodactylum tricornutum. The recombinant mature PtCA1 and -2 (mPtCA1 and -2) were completely inactivated following oxidation by 50 µm CuCl(2), whereas DTT activated CAs in a concentration-dependent manner. The maximum activity of mPtCAs in the presence of 6 mm reduced DTT increased significantly by addition of 10 µm Trxs from Arabidopsis thaliana (AtTrx-f2 and -m2) and 5 µm Trxs from P. tricornutum (PtTrxF and -M). Analyses of mPtCA activation by Trxs in the presence of DTT revealed that the maximum mPtCA1 activity was enhanced ∼3-fold in the presence of Trx, whereas mPtCA2 was only weakly activated by Trxs, and that PtTrxs activate PtCAs more efficiently compared with AtTrxs. Site-directed mutagenesis of potential disulfide-forming cysteines in mPtCA1 and mPtCA2 resulted in a lack of oxidative inactivation of both mPtCAs. These results reveal the first direct evidence of a target of plastidic Trxs in diatoms, indicating that Trxs may participate in the redox control of inorganic carbon flow in the pyrenoid, a focal point of the CO(2)-concentrating mechanism.

The plant ontology as a tool for comparative plant anatomy and genomic analyses.

The Plant Ontology (PO; http://www.plantontology.org/) is a publicly available, collaborative effort to develop and maintain a controlled, structured vocabulary ('ontology') of terms to describe plant anatomy, morphology and the stages of plant development. The goals of the PO are to link (annotate) gene expression and phenotype data to plant structures and stages of plant development, using the data model adopted by the Gene Ontology. From its original design covering only rice, maize and Arabidopsis, the scope of the PO has been expanded to include all green plants. The PO was the first multispecies anatomy ontology developed for the annotation of genes and phenotypes. Also, to our knowledge, it was one of the first biological ontologies that provides translations (via synonyms) in non-English languages such as Japanese and Spanish. As of Release #18 (July 2012), there are about 2.2 million annotations linking PO terms to >110,000 unique data objects representing genes or gene models, proteins, RNAs, germplasm and quantitative trait loci (QTLs) from 22 plant species. In this paper, we focus on the plant anatomical entity branch of the PO, describing the organizing principles, resources available to users and examples of how the PO is integrated into other plant genomics databases and web portals. We also provide two examples of comparative analyses, demonstrating how the ontology structure and PO-annotated data can be used to discover the patterns of expression of the LEAFY (LFY) and terpene synthase (TPS) gene homologs.

WZ4002, a third-generation EGFR inhibitor, can overcome anoikis resistance in EGFR-mutant lung adenocarcinomas more efficiently than Src inhibitors.

Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.

NF-κB signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells.

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cells cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of IκBα, the inhibitor of nuclear factor (NF)-κB, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-κB. Moreover, the inhibition of NF-κB with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-κB signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice.

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.