Mating Pheromone (Gamone 1) in Blepharisma: A Glycoprotein Responsible for Species Diversity in Unicellular Organisms (Alveolata, Ciliophora).

The genus Blepharisma (Alveolata, Ciliophora) is a unicellular organism distributed worldwide, even in extreme environments, and comprises numerous species. While usually proliferating through cell division, Blepharisma undergoes sexual reproduction (conjugation) when cells are moderately starved. Conjugation is initiated by mating pheromones (gamone 1 and gamone 2) secreted by complementary mating-type cells. Gamone 1, a glycoprotein, functions in a species-specific manner, while gamone 2, an amino acid derivative, is a common molecule across species. The specific function of gamone 1 suggests the possibility that mutations in gamone 1 might have led to reproductive isolation during the evolutionary process, triggering species diversification. In this study, by comparing the amino acid sequences of gamone 1 homologs from 15 strains (representing five species), we found that mutations resulting in distinct amino acid properties occur across species boundaries and are mainly concentrated at two specific regions within gamone 1. These mutations potentially alter the binding affinity of gamone 1 to its receptors, suggesting their effect in causing reproductive isolation. The interspecies artificial conjugation conducted previously and the molecular phylogenetic tree constructed using the gamone 1 homolog sequences in this study provide insights into the speciation process within the genus Blepharisma.

Origins of genome-editing excisases as illuminated by the somatic genome of the ciliate Blepharisma.

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one of the two high-level ciliate clades (subphylum Postciliodesmatophora) and, unusually, has dual pathways of somatic nuclear and genome development. This makes it ideal for investigating the functioning and evolution of these processes. Here we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mbp), arranged as numerous telomere-capped minichromosomal isoforms. This genome encodes eight PiggyBac transposase homologs no longer harbored by transposons. All appear subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We hypothesize that PiggyBac homologs were ancestral excisases that enabled the evolution of extensive natural genome editing.

MITE infestation accommodated by genome editing in the germline genome of the ciliate Blepharisma.

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei, a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.

Possible Third Step Preventing Conjugation between Different Species of Blepharisma.

In the genus Blepharisma, reproductive isolation between different species appears to be established at least by two barriers: (1) a mating pheromone, i.e., gamone 1, and (2) a factor involved in pair formation. Using four species, we experimentally investigated other potential barriers to interspecific conjugation in Blepharisma, as well as the first and second barriers. Cell-free fluid from type I cells (CFF1) of B. americanum had no effect on B. undulans, B. japonicum, or B. stoltei. Type II cells of B. americanum responded to CFF1 from B. americanum but not to CFF1 from B. undulans, B. japonicum, or B. stoltei. Gamone 1, therefore, seems to be the first reproductive barrier (with the inclusion of B. americanum species [megakaryotype 3]) as reported previously. In pretreated cells with complementary gamones in B. undulans and B. americanum, inter-species pair formation was rare, but pair formation between B. americanum and B. japonicum and between B. americanum and B. stoltei occurred at relatively high frequency. Most of the inter-species B. americanum−B. stoltei pairs underwent nuclear changes specific to conjugation. No significant difference was observed between the intra- and inter-species pairs over the time course of the nuclear changes, but the percentage of abnormal cells was higher in inter-species pairs than in intra-species pairs, and no progenies were produced by inter-species pairs. These results suggest a third barrier or step, in addition to the first and second ones, in nuclear changes after pair formation that prevents interspecific conjugation in Blepharisma.

A single amino acid residue regulates the substrate affinity and specificity of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyses the oxidative cleavage of L-Trp. The ciliate Blepharisma stoltei has four IDO genes (IDO-I, -II, -III and -IV), which seem to have evolved via two sequential gene duplication events. Each IDO enzyme has a distinct enzymatic property, where IDO-III has a high affinity for L-Trp, whereas the affinity of the other three isoforms for L-Trp is low. IDO-I also exhibits a significant catalytic activity with another indole compound: 5-hydroxy-l-tryptophan (5-HTP). IDO-I is considered to be an enzyme that is involved in the biosynthesis of the 5-HTP-derived mating pheromone, gamone 2. By analysing a series of chimeric enzymes based on extant and predicted ancestral enzymes, we identified Asn131 in IDO-I and Glu132 in IDO-III as the key residues responsible for their high affinity for each specific substrate. These two residues were aligned in an identical position as the substrate-determining residue (SDR). Thus, the substrate affinity and specificity are regulated mostly by a single amino acid residue in the Blepharisma IDO-I and IDO-III enzymes.

Novel Specificity of IDO Enzyme Involved in the Biosynthesis of Mating Pheromone in the Ciliate Blepharisma stoltei.

Mating pheromones (gamone 1 and gamone 2) in the ciliate Blepharisma are biologically active substances that trigger sexual reproduction (conjugation) under starvation conditions. Gamone 1 is a glycoprotein secreted by type I cells, and gamone 2 is a tryptophan (Trp)-derivative compound secreted by type II cells. Both gamones stimulate complementary mating type cells to promote each gamone production and induce pair formation. To elucidate the biosynthetic pathway of gamone 2, we investigated the enzymes involved in the pathway and the specificity of the enzymes. An RNA-seq analysis revealed that Blepharisma stoltei (Heterotrichea) possesses four indoleamine 2,3-dioxygenase (IDO) genes showing distinct expression patterns. Along with results from real-time PCR, these findings demonstrated that each IDO gene has different expression patterns that depend on the cellular conditions. Expression of IDO-I was correlated with the intensity of gamone 2 expression, and the recombinant IDO-I protein showed catalytic activity for 5-hydroxy-L-Trp (5-HTP) but very weak activity for L-Trp. Our results indicate that IDO-I is an enzyme evolutionary specialized to gamone 2 production in Blepharisma, and that the biosynthetic pathway for gamone 2 uses 5-HTP as an intermediate.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

Site-specific migration of human fetal melanocytes in volar skin.

BACKGROUND: Melanocytes originate from the neural crest and migrate ventrally from the dorsal neural tube during embryogenesis. How human melanocytes locate at their suitable positions during embryogenesis, however, is unclear. Although a growing body of evidence indicates that melanocytes, melanoblasts, and melanocyte stem cells are closely related to hair follicles, little is known about volar skin. OBJECTIVE: The aim of this study was to observe skin development during human fetal period and clarify the site-specific migration process of human fetal sole melanocytes. METHODS: We obtained 4-mm punch biopsies from the scalp, back, abdomen, and right sole of 36 aborted fetuses (gestational age 12-21 weeks). We compared the migration process between hairly areas and volar areas by immunohistochemical staining. RESULTS: Immunohistochemical examination revealed that gp100 (HMB-45) sensitively detects human melanocytes in embryogenesis. Melanocytes were present at the epidermal base, where hair placodes/buds form at 12-15 weeks gestation. Fetal melanocytes in hair follicles are supplied from the epidermis. In volar skin, melanocytes originally localize only in the acrosyringium, where they migrate deeper into with gland development at 16-18 weeks gestation. Palmoplantar melanocyte migration and maturation processes differ considerably from those of the other hairy skin sites. CONCLUSION: Eccrine sweat glands seem to have a central role in the palmoplantar melanocyte migration process, similar to the role of hair follicles in hairy sites.

Two possible barriers blocking conjugation between different megakaryotypes of Blepharisma.

We investigated mating pair formation between three Blepharisma species Blepharisma undulans, Blepharisma japonicum, and Blepharisma stoltei to determine whether their respective gamones (mating pheromones) effectively induce mating pairs between different species. Cell-free fluid from type II cells (CFF2) of B. undulans (megakaryotype II) induced pairing of B. japonicum and B. stoltei type I cells (megakaryotype IV), and CFF2 of B. japonicum and B. stoltei induced pairing of B. undulans type I cells. Cell-free fluid from B. undulans type I cells (CFF1) did not induce pairing of B. japonicum and B. stoltei type II cells, and CFF1 of B. japonicum and B. stoltei failed to induce pairing of B. undulans. CFF1 from B. japonicum and B. stoltei mutually induced pairing, as previously reported. These results indicate that gamone 2 is common among megakaryotypes II and IV, and that gamone 1 appears to be at least megakaryotype-specific. When cells belonging to megakaryotypes II and IV are separately pre-treated with effective gamones and mixed, mating pairs between megakaryotypes rarely form. Taken together, these results suggest at least two barriers, a gamone and a factor involved in pair formation, that prevent conjugation between different megakaryotypes of Blepharisma.

Guidelines for obstetrical practice in Japan: Japan Society of Obstetrics and Gynecology (JSOG) and Japan Association of Obstetricians and Gynecologists (JAOG) 2014 edition.

The 'Clinical Guidelines for Obstetrical Practice, 2011 edition' were revised and published as a 2014 edition (in Japanese) in April 2014 by the Japan Society of Obstetrics and Gynecology and the Japan Association of Obstetricians and Gynecologists. The aims of this publication include the determination of current standard care practices for pregnant women in Japan, the widespread use of standard care practices, the enhancement of safety in obstetrical practice, the reduction of burdens associated with medico-legal and medico-economical problems, and a better understanding between pregnant women and maternity-service providers. The number of Clinical Questions and Answers items increased from 87 in the 2011 edition to 104 in the 2014 edition. The Japanese 2014 version included a Discussion, a List of References, and some Tables and Figures following the Answers to the 104 Clinical Questions; these additional sections covered common problems and questions encountered in obstetrical practice, helping Japanese readers to achieve a comprehensive understanding. Each answer with a recommendation level of A, B or C was prepared based principally on 'evidence' or a consensus among Japanese obstetricians in situations where 'evidence' was weak or lacking. Answers with a recommendation level of A or B represent current standard care practices in Japan. All 104 Clinical Questions and Answers items, with the omission of the Discussion, List of References, and Tables and Figures, are presented herein to promote a better understanding among English readers of the current standard care practices for pregnant women in Japan.

Induced pluripotent stem cell generation-associated point mutations arise during the initial stages of the conversion of these cells.

A large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations.

Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.

The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

Alternative gene expression in type I and type II cells may enable further nuclear changes during conjugation of Blepharisma japonicum.

In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis.

Crucial role of c-Myc in the generation of induced pluripotent stem cells.

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.

[Diagnosis and treatment methods in recurrent miscarriage].

Recurrent miscarriage is classically defined as three or more consecutive pregnancy losses. Established causes of recurrent miscarriage are antiphospholipid antibodies, uterine anomalies and abnormal chromosomes in either partner, particularly translocations. Embryonic aneuploidy is the most important cause of miscarriage before ten weeks' gestation. It can be speculated that about 51% of patients with a history of three miscarriages experienced these because of abnormal embryonic karyotypes. It is not necessary to give any medication for such cases caused by an abnormal embryonic karyotype. Psychological support might be the most important requirement to continue conceiving till live birth results.

Glycogen storage disease type Ia (GSD Ia) during pregnancy: report of a case complicated by fetal growth restriction and preeclampsia.

Glycogen storage disease type Ia (GSD Ia) leads to disturbed glycogenolysis and gluconeogenesis due to a deficiency in the enzyme glucose-6-phosphatase. A patient with GSD Ia showed hypoglycemia and proteinuria without dietary management since early pregnancy. The patient's condition was complicated by hypertension with increase in proteinuria at 22 weeks of gestation. In spite of administration of antihypertensive drugs and dietary management, the disease became more severe with deterioration in the fetal status and inhibition of fetal growth. Thus, a cesarean section was performed at 26 weeks of gestation. The delivered male infant weighing 412 g died at 2 days after birth. The patient's blood pressure had normalized within 3 months after delivery, while proteinuria persisted.

Behavioural changes induced by the conjugation-inducing pheromones, gamone 1 and 2, in the ciliate Blepharisma japonicum.

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.

Synthesis of fluorescent molecular probes specific for the receptor of blepharismone, a mating-inducing pheromone of the ciliate Blepharisma japonicum.

Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molecular probes for the gamone 2 receptor. Further, we proved that they have inhibitory activities against the blepharismone-induced monotypic pairing of B. japonicum.

Pregnancy and delivery in protein C-deficiency.

Activated protein C (APC) is a strong inhibitor of coagulation, inactivating coagulation factors Va and VIIIa upon binding to protein S (PS) in the presence of thrombin and thrombomodulin. The normal concentration of PC in the plasma is approximately 4 microg/ml. Throughout pregnancy, PC activity and antigenic levels show no significant trend and remain within the normal reference range. Several PC point mutations have been documented, including those characterized as type I and II PC deficiencies. These, as well as mutations in Protein S (PS), factors Va and VIIIa, and thrombomodulin, can result in venous thromboses of various degrees of severity. The relative risk of thrombosis with PC deficiency is 7.3%. In pregnancy, the risk is 3-10% antepartum and 7-19% postpartum. PC deficiency has also been reported to be associated with both non-recurrent and recurrent first, second and third trimester miscarriages, intrauterine fetal death, intrauterine growth retardation, placental abruption and preeclampsia. However, prior to 10 weeks of gestation, no significant relationship between PC deficiency and pregnancy loss has been established.