Aneuploid rescue precedes X-chromosome inactivation and increases the incidence of its skewness by reducing the size of the embryonic progenitor cell pool.

STUDY QUESTION: Do monosomy rescue (MR) and trisomy rescue (TR) in preimplantation human embryos affect other developmental processes, such as X-chromosome inactivation (XCI)? SUMMARY ANSWER: Aneuploid rescue precedes XCI and increases the incidence of XCI skewness by reducing the size of the embryonic progenitor cell pools. WHAT IS KNOWN ALREADY: More than half of preimplantation human embryos harbor aneuploid cells, some of which can be spontaneously corrected through MR or TR. XCI in females is an indispensable process, which is predicted to start at the early-blastocyst phase. STUDY DESIGN, SIZE, DURATION: We examined the frequency of XCI skewness in young females who carried full uniparental disomy (UPD) resulting from MR or TR/gamete complementation (GC). The results were statistically analyzed using a theoretical model in which XCI involves various numbers of embryonic progenitor cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 39 children and young adults ascertained by imprinting disorders. XCI ratios were determined by DNA methylation analysis of a polymorphic locus in the androgen receptor gene. We used Bayesian approach to assess the probability of the occurrence of extreme XCI skewness in the MR and TR/GC groups using a theoretical model of 1-12 cell pools. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 12 of 39 individuals (31%) showed skewed XCI. Extreme skewness was observed in 3 of 15 MR cases (20%) and 1 of 24 TR/GC cases (4.2%). Statistical analysis indicated that XCI in the MR group was likely to have occurred when the blastocyst contained three or four euploid embryonic progenitor cells. The estimated size of the embryonic progenitor cell pools was approximately one-third or one-fourth of the predicted size of normal embryos. The TR/GC group likely had a larger pool size at the onset of XCI, although the results remained inconclusive. LIMITATIONS, REASONS FOR CAUTION: This is an observational study and needs to be validated by experimental analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study provides evidence that the onset of XCI is determined by an intrinsic clock, irrespectively of the number of embryonic progenitor cells. Our findings can also be applied to individuals without UPD or imprinting disorders. This study provides a clue to understand chromosomal and cellular dynamics in the first few days of human development, their effects on XCI skewing and the possible implications for the expression of X-linked diseases in females. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Grants-in-aid for Scientific Research on Innovative Areas (17H06428) and for Scientific Research (B) (17H03616) from Japan Society for the Promotion of Science (JSPS), and grants from Japan Agency for Medical Research and Development (AMED) (18ek0109266h0002 and 18ek0109278h0002), National Center for Child Health and Development and Takeda Science Foundation. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Superconducting spin smecticity evidencing the Fulde-Ferrell-Larkin-Ovchinnikov state in Sr2RuO4.

Translational symmetry breaking is antagonistic to static fluidity but can be realized in superconductors, which host a quantum-mechanical coherent fluid formed by electron pairs. A peculiar example of such a state is the Fulde-Ferrell-Larkin-Ovchinnikov (FFLO) state, induced by a time-reversal symmetry-breaking magnetic field applied to spin-singlet superconductors. This state is intrinsically accompanied by the superconducting spin smecticity, spin density-modulated fluidity with spontaneous translational-symmetry breaking. Detection of such spin smecticity provides unambiguous evidence for the FFLO state, but its observation has been challenging. Here, we report the characteristic "double-horn" nuclear magnetic resonance spectrum in the layered superconductor Sr2RuO4 near its upper critical field, indicating the spatial sinusoidal modulation of spin density that is consistent with superconducting spin smecticity. Our work reveals that Sr2RuO4 provides a versatile platform for studying FFLO physics.

Cryptococcal pleuritis developing in a patient on regular hemodialysis.

A 64-year-old male on regular hemodialysis who was a human T lymphotrophic virus Type I (HTLV-I) carrier developed cryptococcal pleuritis. The initial manifestations of the present case were a persistent cough and the accumulation of unilateral pleural effusion. A culture of the pleural fluid of the patient grew cryptococcus neoformans and a test for antigens against cryptococcus neoformans in the pleural fluid was also positive, therefore, cryptococcal pleuritis was diagnosed. Pleural cryptococcosis per se is rare and it is extremely rare for a dialysis patient to develop pleural cryptococcosis. To our knowledge, only a few cases of cryptococcal pleuritis have so far been reported in patients on dialysis. Furthermore, an isolated occurrence of cryptococcal pleuritis with no cryptococcal pulmonary parenchymal lesions, as was seen in the present case, is rare because cryptococcal pleuritis is usually associated with underlying cryptococcal pulmonary parenchymal lesions. Patients on chronic dialysis are susceptible to developing pleural effusion from many etiologies such as congestive heart failure, infection (tuberculosis, bacterial, viral, parasitic, fungal), collagen vascular disease, drug reaction, metastasis, or uremia itself. Cryptococcal pleuritis developing in a dialysis patient is extremely rare, but physicians should consider cryptococcal infection as a possible cause when pleural effusion develops in a dialysis patient and no other cause is identified, as occurred in the present case.

Arsenic trioxide (As2O3)-induced apoptosis and differentiation in retinoic acid-resistant acute promyelocytic leukemia model in hGM-CSF-producing transgenic SCID mice.

Recent clinical studies in China and USA showed that arsenic trioxide (As2O3) is an effective treatment of acute promyelocytic leukemia (APL) patients refractory to all-trans retinoic acid (RA). We here investigate the effects of As2O3 on RA-resistant APL in vivo and in vitro using our RA-resistant APL model system. As2O3 can induce inhibition of cellular growth of both RA-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis in vitro. The expression of BCL-2 protein decreased in a dose- and time-dependent manner in NB4 cells. Interestingly, the levels of BCL-2 protein were not modulated by As2O3, but it did upregulate BAX protein in UF-1 cells. UF-1 cells (1x10(7)) were transplanted into hGM-CSF-producing transgenic SCID mice and successfully formed subcutaneous tumors. After 40 days of implantation, mice were treated with As2O3, all-trans RA and PBS for 21 days. In all-trans RA- and PBS-treated mice, tumors grew rapidly, with a 4.5-fold increase in volume at day 21 compared to the initial size. In marked contrast, tumor size was decreased to half of the initial size by the treatment of As2O3, which resulted in cells with the typical appearance of apoptosis. Interestingly, one of the As2O3-treated mice showed mature granulocytes in the diminished tumor, suggesting that As2O3 had dual effects on RA-resistant APL cells in vivo: both inducing apoptosis and differentiation of the leukemic cells. We conclude that our RA-resistant APL model will be useful for evaluating novel therapeutic approaches to patients with RA-resistant APL, and for further investigation of the metabolism of As2O3 in vivo.

Novel mutation in the PML/RARalpha chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia.

OBJECTIVE: All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS: We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS: A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS: These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.

Thrombin inhibitors. 1. Ester derivatives of N alpha-(arylsulfonyl)-L-arginine.

A series of N alpha-(arylsulfonyl)-L-arginine esters was prepared and tested as inhibitors of the clotting activity of thrombin. N alpha-Dansyl-L-arginine methyl ester was the most inhibitory of the N alpha-(arylsulfonyl)-L-arginine methyl esters. The most potent inhibitors were the n-propyl and n-butyl esters of N alpha-dansyl-L-arginine with an I50 of 2 X 10(-6) M. Esters of unsaturated straight-chain alcohols with a chain length of four carbons were also as inhibitory as the n-butyl ester. The inhibitors were hydrolyzed by thrombin and trypsin more slowly than N alpha-tosyl-L-arginine methyl ester.

Purification and some chemical properties of thrombin-like enzyme from Trimeresurus flavoviridis venom.

There has been no previous report indicating whether thrombin-like enzyme is contained in the venom of Trimeresurus flavoviridis which has the strongest toxic effect in the case of Habu bite. The present study was undertaken to clarify the existence of thrombin-like enzyme in Trimeresurus flavoviridis venom. As a starting material, lyophilized crude venom of Trimeresurus flavoviridis was used, and ammonium sulphate fractionation, gel filtration using Sephadex G-25, Sephadex G-150 and arginine-Sepharose affinity chromatography were carried out to separate and purify a thrombin-like enzyme from the crude venom. The enzyme was purified to a 137-fold increase in specific activity and the purified preparation revealed a single band on SDS-PAGE. The molecular weight of the enzyme was estimated to be 65,000-70,000 daltons by means of SDS-PAGE and gel filtration, and its isoelectric point was pH 4.5-5.5. Furthermore, the optimal pH of the enzyme was in the range of pH 8.0 to 8.5. Some of the differences in enzymatic properties between this enzyme and bovine thrombin were studied. The snake enzyme could coagulate only rabbit plasma and convert only purified rabbit fibrinogen to fibrin gel. In addition, this thrombin-like enzyme released only fibrinopeptide A from purified rabbit fibrinogen and did not release fibrinopeptide B.

Minor daphnane-type diterpenoids from Wikstroemia retusa.

In addition to huratoxin, pimelea factor P2, and wikstroelides A-G from the fresh bark, eight daphnane-type diterpenoids, wikstroelides H-O, were isolated from the bark and stem, and their structures established. Cytotoxicity was assayed on some wikstroelides.

Interaction of fibrinolytic activity between the tracheobronchial secretion and circulating blood of rats.

In order to clarify the origin and release mechanism of plasminogen activator in the tracheobronchial secretion of rats, electrophoretic analysis of the secretion and studies on the effects of certain vasoactive drugs on the activator activity in the secretion were carried out. From the results of electrophoretic analysis of the tracheobronchial secretion in non-treated rats, protease inhibitor composed of glycoprotein was not contained in the secretion in contrast to the circulating blood, but a protein of low molecular weight like albumin in the circulating blood was contained in the secretion. Furthermore, after injection of noradrenalin, the blood pressure was temporarily elevated and the fibrinolytic activity of the euglobulin fraction in the circulatory blood was also increased. Subsequent to this elevation of fibrinolytic activity in the circulating blood, the fibrinolytic activity in the tracheobronchial secretion increased. Based on these results, it is suggested that the increase of fibrinolytic activity in the circulating blood was due to increased release of plasminogen activator from the vascular wall, and the increased fibrinolytic activity of the tracheobronchial secretion was caused by a consequent increased transudation of plasminogen activator from the circulating blood into the tracheobronchial lumen.

Role of the autonomic nervous system in regulating salivary mucin secretion by the canine submandibular gland in vivo.

The secretion of mucin was assessed by measuring changes in protein and sialic acid concentrations in saliva. Electrical stimulation of the sympathetic nerve increased protein and sialic acid concentrations greatly but stimulation of the parasympathetic nerve by bethanechol (5-20 micrograms/kg, intravenously) caused a slight increase. Sympathomimetic drugs (1-10 micrograms/kg, intravenously) also increased protein and sialic acid concentrations (isoproterenol greater than adrenaline greater than noradrenaline). Mucin secretion was increased by H133/22, a beta 1 adrenoceptor agonist, or terbutaline, which is roughly equipotent. Adrenaline-induced mucin secretion was inhibited by propranolol, but not by tolazoline. These data suggest that mucin secretion is predominantly regulated by the sympathetic nervous system via beta adrenoceptors, both beta 1 and beta 2.

Demographic study of first-ever stroke and acute myocardial infarction in Okinawa, Japan.

We performed a cross-sectional survey of stroke and acute myocardial infarction (AMI) in Okinawa, Japan with a census population of about 1.2 million. A total of 3,644 cases of first-ever stroke and 898 cases of initial acute myocardial infarction (AMI) were detected. The age-adjusted annual incidence rate for stroke was 105 per 100,000 standard population of Japan, and that of AMI was 26. The case-fatality rate of stroke within 28 days of onset was 12.8%, and that of AMI was 22.2%. Of the stroke cases, 51.4% were diagnosed as brain infarction, 38.7% as brain hemorrhage, and 9.3% as subarachnoid hemorrhage. The diagnosis of stroke subtypes were confirmed by computed tomography or magnetic resonance imaging in 98.4% of all stroke cases. In Okinawa, the incidence rate of AMI was still considerably lower than that in the Western population, and the rate of stroke was similar to that in the Western population.

Studies on proactivator from the paranasal mucous membrane in chronic sinusitis.

We succeeded in differentiating the proactivator (PA) in tissue extract of paranasal mucous membrane with chronic sinusitis by the gel filtration technique. Furthermore, the results demonstrated that PA in the tissue extracts of the paranasal mucous membrane with chronic sinusitis and antrochoanal polyp was unrelated to the antigenicity of plasminogen. In particular, it was clarified that the tissue extract of antrochoanal polyp as a source of PA was not related to the antigenicity of plasminogen.

The role of fibrinolytic enzymes in inflammation and paranasal mucous membrane.

The distribution and role of tissue plasminogen activator (TA) and proactivator (PA) in various diseases of the nasal and paranasal cavity were investigated. The stronger the inflammatory and proliferative response of the paranasal mucous membrane, the weaker was the fibrinolytic activity of TA. The fibrinolytic activity of PA tended to be stronger than TA activity. It is considered that PA may play an important role in inflammatory enlargement and proliferation of the paranasal mucous membrane, but does not play an important role in carcinogenic enlargement and proliferation of the nasal and paranasal mucous membrane.

Control of Arthus tonsillitis by the administration of an antiplasminic agent.

Previous reports have shown that fibrinolytic activity was increased in the circulatory blood of rabbits with Arthus tonsillitis. In this study, t-AMCHA was administered intravenously to rabbits, in order to clarify its role as an antiplasminic agent in the control of Arthus tonsillitis. First, to establish the proper method of administration of t-AMCHA and the quantity to be administered, a preliminary estimation was made of the requisite concentration of t-AMCHA in the circulating blood and tonsillar tissue. Then macroscopic observations and histopathological studies were performed on the tonsils of rabbits with Arthus tonsillitis after t-AMCHA administration at various doses. Finally, after injection of the antiplasminic agent, estimations were made of the plasma fibrinolytic activity in the blood of the rabbits under study. It was decided that administration of t-AMCHA at 3-hourly intervals could maintain a constant concentration of the substance in the tonsillar tissue. The marked hyperaemia and bleeding which were observed macroscopically and histopathologically at 1-2 days after the onset of tonsillitis, disappeared from the parenchyma of the tonsils and pharyngeal tissue surrounding the tonsils in the group receiving regular injections of t-AMCHA at 3-hourly intervals. After estimating certain parameters of the fibrinolytic system in the blood, it was found that fibrinolytic activity decreased and whole plasmin was not consumed as a result of the administration of t-AMCHA during the early stage of tonsillitis. The process of Arthus tonsillitis can thus be controlled by the administration of the antiplasminic agent.

Antiplasmin activity in carrageenin-induced inflammation of rats.

The significance of inhibitors in the coagulation-fibrinolysis system was examined by inducing experimental inflammation with carrageenin. Fast-acting plasmin inhibitor, alpha 2-plasmin inhibitor (alpha 2-Pl), was recently discovered in human blood and it was stated that such alpha 2-Pl represents the most sensitive and specific inhibitor of plasmin. The possible role of alpha 2-Pl has been studied in detail in vitro and in vivo in relation to the thrombolytic condition and bleeding based on hyperfibrinolysis. However, the significance of alpha 2-Pl has not been clarified for the inflammatory state and diseases. In the present study in order to clarify the possible role of alpha 2-Pl in inflammatory responses, the antiplasmin activity in the plasma and local fluid (exudate) was investigated using an animal model of inflammation. After subcutaneous injection of carrageenin into the dorsum of rats, on day 7 and 21, the antiplasmin activity in the plasma was significantly higher than that in the plasma of non-treated rats (p less than 0.001). However, the antiplasmin activity in the plasma on day 7 was not significantly different from that on day 21. On the other hand, the antiplasmin activity in the exudate on day 21 was significantly lower than that on day 7 (p less than 0.001). Based on ;these results, it is suggested that the antiplasmin in the inflammatory exudate was consumed during the inflammation to form the hard wall of the carrageenin pouch.

Differentiation of proactivator from tissue plasminogen activator with low molecular weight in the paranasal mucous membrane.

It has been shown that large amounts of fibrinolytic enzyme are contained in tissue extracts of the paranasal mucous membrane in patients with chronic sinusitis. However, the fibrinolytic enzyme in the tissue extracts has not yet been characterized by biochemical techniques. In the present study, proactivator was differentiated from tissue plasminogen activator with low molecular weight in extracts of the paranasal mucous membrane and the existence of the proactivator was thus demonstrated in tissue extracts. By analogy with the proactivator in antrochoanal polyps, this proactivator may play an important role in the proliferation and enlargement of the paranasal mucous membrane.

The detection of fibrinogen (or FgDP)-protamine sulphate complex in vitro and in the circulating blood.

In order to clarify the pathophysiological significance of the fibrinogen (FgDP)-protamine complex, the interaction between fibrinogen and protamine sulphate was studied in vitro and in vivo. Using the electrophoretic technique, an attempt was made to determine whether the complex could be detected in vitro and in the circulating blood. From the present studies, it has been clarified in in vitro experiments that the slower mobility of two peaks on immunoelectrophoresis represented a complex of protamine sulphate and fibrinogen (FgDP). Furthermore, the peak of slower mobility on crossed immunoelectrophoresis appeared in the circulating blood of a rabbit receiving administration of excess protamine sulphate. That is, in vivo experiments showed that a complex of protamine sulphate and fibrinogen (FgDP) could be detected in the circulating blood.