Essential role of mouse Dead end1 in the maintenance of spermatogonia.

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of Asingle and Apaired spermatogonia were preferentially decreased as compared with those of Aaligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development.

Different properties of polysialic acids synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4.

Polysialic acid (polySia) is mainly found as a modification of neural cell adhesion molecule (NCAM) in whole embryonic brains, as well as restricted areas of adult vertebrate brains, including the hippocampus. PolySia shows not only repulsive effects on NCAM-involved cell-cell interactions due to its bulky and hydrated properties, but also attractive effects on the interaction with neurologically active molecules, which exerts a reservoir function. Two different polysialyltransferases, ST8SIA2 and ST8SIA4, are involved in the synthesis of polySia chains; however, to date, the differences of the properties between polySia chains synthesized by these two enzymes remain unknown. In this study, to clarify this point, we first prepared polySia-NCAMs from HEK293 cells stably expressing ST8SIA4 and ST8SIA2, or ST8SIA2 (SNP-7), a mutant ST8SIA2 derived from a schizophrenia patient. The conventional sensitive chemical and immunological characterizations showed that the quantity and quality (structural features) of polySia are not so much different between ST8SIA4- and ST8SIA2-synthesized ones, apart from those of ST8SIA2 (SNP-7). Then, we assessed the homophilic and heterophilic interactions mediated by polySia-NCAM by adopting a surface plasmon resonance measurement as an in vitro analytical method. Our novel findings are as follows: (i) the ST8SIA2- and ST8SIA4-synthesized polySia-NCAMs exhibited different attractive and repulsive effects than each other; (ii) both polySia- and oligoSia-NCAMs synthesized by ST8SIA2 were able to bind polySia-NCAMs; (iii) the polySia-NCAM synthesized by a ST8SIA2 (SNP-7) showed markedly altered attractive and repulsive properties. Collectively, polySia-NCAM is suggested to simultaneously possess both attractive and repulsive properties that are highly regulated by the two polysialyltransferases.

Chlorpromazine Increases the Expression of Polysialic Acid (PolySia) in Human Neuroblastoma Cells and Mouse Prefrontal Cortex.

The neural cell adhesion molecule (NCAM) is modified by polysialic acid (polySia or PSA) in embryonic brains. In adult brains, polySia modification of NCAM is only observed in restricted areas where neural plasticity, remodeling of neural connections, or neural generation is ongoing although the amount of NCAM remains unchanged. Impairments of the polySia-expression and several single nucleotide polymorphisms (SNPs) of the polysialyltransferase (polyST) ST8SIA2 gene are reported to be associated with schizophrenia and bipolar disorder. Chlorpromazine (CPZ) is well-known as an agent for treating schizophrenia, and our hypothesis is that CPZ may affect the polySia expression or the gene expression of polySTs or NCAM. To test this hypothesis, we analyzed the effects of CPZ on the expression of polySia-NCAM on human neuroblastoma cell line, IMR-32 cells, by immunochemical and chemical methods. Interestingly, the cell surface expression of polySia, especially those with lower chain lengths, was significantly increased on the CPZ-treated cells, while mRNAs for polySTs and NCAM, and the amounts of total polySia-NCAM remained unchanged. The addition of brefeldin A, an inhibitor of endocytosis, suppressed the CPZ-induced cell surface polySia expression. In addition, polySia-NCAM was also observed in the vesicle compartment inside the cell. All these data suggest that the level of cell surface expression of polySia in IMR-32 is highly regulated and that CPZ changes the rate of the recycling of polySia-NCAM, leading to the up-regulation of polySia-NCAM on the cell surface. We also analyzed the effect of CPZ on polySia-expression in various brain regions in adult mice and found that CPZ only influenced the total amounts of polySia-NCAM in prefrontal cortex. These results suggest a brain-region-specific effect of CPZ on the expression of total polySia in mouse brain. Collectively, anti-schizophrenia agent CPZ consistently up-regulates the expression polySia at both cellular and animal levels.

Arterial blood pressure response to head-up tilt test and orthostatic tolerance in nurses.

OBJECTIVES: High tolerance to postural changes was examined in nurses. METHODS: Twelve female nurses and 12 healthy controls underwent a 70° head-up tilt (HUT) test for 10 min. Blood pressure (BP), heart rate (HR), pulse pressure, and hormone levels were measured. Baroreceptor sensitivity (BRS) was calculated using a sequence technique. RESULTS: HR increased during HUT in both subject groups, with no difference between groups. Systolic BP was rapidly increased by HUT in both subject groups, and was higher in the nurse group than in the control group during the first 2 min of HUT. Pulse pressure decreased during 1-2.5 min of HUT in the control group, but there was no decrease in the nurse group. BRS was decreased by HUT in the nurse group, while it tended to be decreased in the control group. Both during baseline and HUT, BRS was lower in the nurse group than in the control group. Plasma noradrenaline increased with HUT, and the increase was greater in the nurse group than in the control group. CONCLUSIONS: Although nurse subjects had a lower BRS during HUT than control subjects, they were able to effectively maintain BP during HUT, suggesting that nurse subjects had higher orthostatic tolerance. The better maintenance of BP in nurse subjects appeared to be associated with a compensatory mechanism other than the arterial baroreflex and/or a hemodynamic mechanism.

Loss of Dead end1 induces testicular teratomas from primordial germ cells that failed to undergo sexual differentiation in embryonic testes.

Spontaneous testicular teratomas (STTs) are tumours comprising a diverse array of cell and tissue types, which are derived from pluripotent stem-like cells called embryonal carcinoma cells (ECCs). Although mouse ECCs originate from primordial germ cells (PGCs) in embryonic testes, the molecular basis underlying ECC development remains unclear. This study shows that the conditional deletion of mouse Dead end1 (Dnd1) from migrating PGCs leads to STT development. In Dnd1-conditional knockout (Dnd1-cKO) embryos, PGCs colonise the embryonic testes but fail to undergo sexual differentiation; subsequently, ECCs develop from a portion of the PGCs. Transcriptomic analyses reveal that PGCs not only fail to undergo sexual differentiation but are also prone to transformation into ECCs by upregulating the expression of marker genes for primed pluripotency in the testes of Dnd1-cKO embryos. Thus, our results clarify the role of Dnd1 in developing STTs and developmental process of ECC from PGC, providing novel insights into pathogenic mechanisms of STTs.

CSF1R mutations identified in three families with autosomal dominantly inherited leukoencephalopathy.

Genetic and phenotypic heterogeneities are considerably high in adult-onset leukoencephalopathy, in which comprehensive mutational analyses of the candidate genes by conventional methods are too laborious. We applied exome sequencing to conduct a comprehensive mutational analysis of genes for autosomal dominant leukoencephalopathies. Genomic DNA samples from four patients of three families with autosomal dominantly inherited adult-onset leukodystrophy were subjected to exome sequencing. On the basis of the results, 21 patients with adult-onset sporadic leukodystrophy and one patient with pathologically proven HDLS were additionally screened for CSF1R mutations. Exome sequencing identified heterozygous CSF1R mutations (p.I794T and p.R777W) in two families. I794T has recently been reported as a causative mutation for hereditary diffuse leukoencephalopathy with spheroids (HDLS), and R777W is a novel mutation. Although mutational analysis of CSF1R in 21 sporadic cases revealed no mutations, another novel CSF1R mutation, p.C653Y, was identified in one patient with autopsy-proven HDSL. These variants were located in the PTK domain where the causative mutations cluster. Functional prediction of the mutant CSF1R as well as cross-species conservation of the affected amino acids supports the notion that these variants are pathogenic for HDLS. Exome sequencing is useful for a comprehensive mutational analysis of causative genes for hereditary leukoencephalopathies, and CSF1R should be considered a candidate gene for patients with autosomal dominant leukoencephalopathies.

Selective stimulation of nociceptive small fibers during intraepidermal electrical stimulation: Experiment and computational analysis.

Electrical stimulation of skin nociceptors is gaining attention in pain research and peripheral neuropathy diagnosis. However, the optimal parameters for selective stimulation are still difficult to determine because they require simultaneous characterization of the electrical response of small fibers (Aδ- and C-fibers). In this study, we measured the in vivo electrical threshold responses of small fibers to train-pulse stimulation in humans for the first time. We also examined selective stimulation via a computational model, which combines electrical analysis, and terminal fiber and synaptic models, including the first cutaneous pain C-fiber model. Selective stimulation of small fibers is performed by injecting train-pulse stimulation via coaxial electrodes with an intraepidermal needle tip at varying pulse counts and frequencies. The activation Aδ- or C-fibers was discriminated from the differences in reaction time. Aδ-fiber elicited a pinpricking sensation with a mean reaction time of 0.522 s, and C-fiber elicited a tingling sensation or slight burning itch with a mean reaction time of 1.243 s. The implemented multiscale electrical model investigates synaptic effects while considering stimulation waveform characteristics. Experimental results showed that perception thresholds decreased with the number of consecutive pulses and frequency up to convergence (five pulses or 70 Hz) during the selective stimulation of Aδ- and C-fibers. Considering the synaptic properties, the optimal stimulus conditions for selective stimulation of Aδ- vs. C-fibers were train of at least four pulses and a frequency of 40-70 Hz at a pulse width of 1 ms. The experimental results were modeled with high fidelity by incorporating temporal synaptic effects into the computational model. Numerical analysis revealed terminal axon thickness to be the most important biophysical factor affecting threshold variability. The computational model can be used to estimate perception thresholds while understanding the mechanisms underlying the selective stimulation of small fibers. The parameters derived here are important in exploring selective stimulation between Aδ- and C-fibers for diagnosing neuropathies.

Estimation of mRNA COVID-19 Vaccination Effectiveness in Tokyo for Omicron Variants BA.2 and BA.5: Effect of Social Behavior.

The variability of the COVID-19 vaccination effectiveness (VE) should be assessed with a resolution of a few days, assuming that the VE is influenced by public behavior and social activity. Here, the VE for the Omicron variants (BA.2 and BA.5) is numerically derived for Japan's population for the second and third vaccination doses. We then evaluated the daily VE variation due to social behavior from the daily data reports in Tokyo. The VE for the Omicron variants (BA.1, BA.2, and BA.5) are derived from the data of Japan and Tokyo with a computational approach. In addition, the effect of the different parameters regarding human behavior on VE was assessed using daily data in Tokyo. The individual VE for the Omicron BA.2 in Japan was 61% (95% CI: 57-65%) for the second dose of the vaccination from our computation, whereas that for the third dose was 86% (95% CI: 84-88%). The individual BA.5 VE for the second and third doses are 37% (95% CI: 33-40%) and 63% (95% CI: 61-65%). The reduction in the daily VE from the estimated value was closely correlated to the number of tweets related to social gatherings on Twitter. The number of tweets considered here would be one of the new candidates for VE evaluation and surveillance affecting the viral transmission.

Mouse dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence.

Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism.

[Relationship between cardiac 123I-metaiodobenzylguanidine scintigraphy and cardiovascular autonomic function test (standing test) in Parkinson's disease].

We investigated the correlation between results of 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy and those of cardiovascular autonomic function tests in patients with Parkinson's disease (PD). 123I-MIBG myocardial scintigraphy and a 5-minute standing test were performed in 50 patients with PD and in 19 control subjects. The value of the basal plasma noradrenaline (NA) level was used as an index of basal sympathetic nerve activity, and %NA was used to assess the response of sympathetic nerve activity. In addition, the parameters of DeltaBP and DeltaHR were evaluated to assess the autonomic response of the cardiovascular system. A mild, but significant correlation was observed between the myocardium to mediastinum (H/M) ratio and the values of the plasma NA baseline (r = 0.35, p < 0.05 in early image, r = 0.29, p < 0.05 in delay image). No significant correlation was observed between the H/M ratio and the other parameters (%NA, DeltaBP, DeltaHR). These results suggest that 123I-MIBG myocardial scintigraphy may be associated with the basal sympathetic nerve activity, but not with autonomic nervous response of the cardiovascular system in patients with PD.

Syncopal attack alters the burst properties of muscle sympathetic nerve activity in humans.

This study aimed at examining whether the properties of microneurographically recorded muscle sympathetic nerve activity (MSNA) were altered during hypotensive attacks. A retrospective study was performed on 74 subjects who participated in tilt studies when vasodepressive syncope was induced incidentally in six subjects. The specific features of MSNA that distinguish this activity from skin sympathetic nerve activity are (1) rhythmic pulse synchronous burst discharge, (2) a duration of approximately 150-300 ms, and (3) no response to arousal stimuli were abolished during the syncopal attack. The altered features observed during the syncopal attack in these six subjects were (1) scattered reflex latencies of MSNA peak from the ECG R-wave, (2) elongated burst duration twice to five times as long as that in conscious state, and (3) response to arousal stimuli. The reduced input from the baroreceptors due to suppression on the central sympathetic volley proximal to the nucleus tractus solitarius might be attributed to the lost features characteristic of MSNA.

Age-related changes in vasomotor reflex control of calf venous capacitance response to lower body negative pressure in humans.

The present study was performed to test the hypothesis that calf venous capacitance would be reduced by mild gravitational stress through a vasomotor reflex in humans, and this response could be diminished with advancing age. Nine young (31 +/- 1 years, mean +/- SE) and 9 elderly (69 +/- 1 years) healthy males were exposed to a lower body negative pressure (LBNP) of 15 mmHg. Venous occlusion plethysmography was used to measure calf venous capacitance and calf blood flow. Muscle sympathetic nerve activity (MSNA) was recorded microneurographically from the tibial nerve along with cardiovascular variables. It was found that baseline MSNA was higher [21 +/- 4 (mean +/- SE) vs. 37 +/- 5 bursts x min(-1), young vs. elderly; p < 0.05] and calf venous capacitance was lower (1.71 +/- 0.12 vs. 1.44 +/- 0.10, ml x 100 ml(-1), young vs. elderly; p < 0.05) in the elderly group. At 15 mmHg-LBNP, heart rate and mean arterial pressure both remained unchanged, MSNA was enhanced, and calf blood flow was reduced in all subjects. Calf venous capacitance during LBNP decreased in the young, but did not change in the elderly. A significant negative correlation between percent changes in MSNA and percent changes in calf venous capacitance existed in the young group (y = 20.171x-11.863, r = 20.682; p = 0.0432), but disappeared in the elderly group. The ratio of percent changes in calf venous capacitance to percent changes in MSNA was markedly lower in the elderly (p < 0.01). In conclusion, these results substantiate our hypothesis that calf venous capacitance is reduced by mild LBNP through the vasomotor reflex, and this response is diminished in the elderly.

Age-related influences of leg vein filling and emptying on blood volume redistribution and sympathetic reflex during lower body negative pressure in humans.

To test the hypothesis that leg vein filling and emptying functions could be impaired with advancing age, which would produce less blood volume redistribution toward the lower body and smaller sympathetic reflex response during mild gravitational stress, 9 young and 10 elderly healthy males were exposed to a lower body negative pressure (LBNP) of 15 mmHg. Venous occlusion plethysmography was used to determine the functions of the leg veins. We found that the baseline venous distensibility index (VDI) was lower (0.057 +/- 0.004 vs. 0.048 +/- 0.003 ml x 100 ml(-1) x mmHg(-1), young vs. elderly; p < 0.05), and half-emptying time (T(1/2)) was shorter (1.6 +/- 0.1 vs. 1.3 +/- 0.1 s, young vs. elderly; p < 0.05) in the elderly. At 15 mmHg-LBNP, VDI was decreased and T(1/2) was shortened significantly in the young group, but only slightly in the elderly group. Neither blood pressure nor heart rate changed significantly in either group. The reduction in peripheral venous pressure, which was recorded from the left antecubital vein at the cubital fossa, was less in the elderly, indicating a smaller decrease in central blood volume during LBNP; however, the enhancement of muscle sympathetic nerve activity was nearly the same as that in the young. We conclude that leg vein filling and emptying functions are impaired in elderly people, producing less blood pooling in the legs and smaller reduction in peripheral venous pressure during LBNP; the maintained sympathetic reflex response might be attributable to the well-preserved baroreflex function control of sympathetic outflow to the muscle in the elderly.

Interaction of NANOS2 and NANOS3 with different components of the CNOT complex may contribute to the functional differences in mouse male germ cells.

NANOS2 and NANOS3 belong to the Nanos family of proteins that contain a conserved zinc finger domain, which consists of two consecutive CCHC-type zinc finger motifs, and contribute to germ cell development in mice. Previous studies indicate that there are redundant and distinct functions of these two proteins. NANOS2 rescues NANOS3 functions in the maintenance of primordial germ cells, whereas NANOS3 fails to replace NANOS2 functions in the male germ cell pathway. However, the lack of a conditional allele of Nanos3 has hampered delineation of each contribution of NANOS2 and NANOS3 to the male germ cell pathway. In addition, the molecular mechanism underlying the distinct functions of these proteins remains unexplored. Here, we report an unexpected observation of a transgenic mouse line expressing a NANOS2 variant harboring mutations in the zinc finger domain. Transcription of Nanos2 and Nanos3 was strongly compromised in the presence of this transgene, which resulted in the mimicking of the Nanos2/Nanos3 double-null condition in the male gonad. In these transgenic mice, P-bodies involved in RNA metabolism had disappeared and germ cell differentiation was more severely affected than that in Nanos2-null mice, indicating that NANOS3 partially substitutes for NANOS2 functions. In addition, similar to NANOS2, we found that NANOS3 associated with the CCR4-NOT deadenylation complex but via a direct interaction with CNOT8, unlike CNOT1 in the case of NANOS2. This alternate interaction might account for the molecular basis of the functional redundancy and differences in NANOS2 and NANOS3 functions.

Forearm elevation augments sympathetic activation during handgrip exercise in humans.

Although angina pectoris in patients with coronary heart disease often occurs when their forearms are in an elevated position for a prolonged period, and sympathetic activation is a major cause of this condition, little is known about the physiological effects of forearm elevation on sympathetic activity during forearm exercise. We hypothesized that forearm elevation augments sympathetic activation during the static handgrip exercise in humans. A total of 10 healthy male volunteers performed 2 min of static handgrip exercise at 30% of maximal voluntary contraction followed by 2 min of post-exercise muscle ischaemia (PEMI; specific activation of the muscle metaboreflex) with two forearm positions: the exercising forearm was elevated 50 cm above the heart (forearm-elevated trial) or fixed at the level of the heart (heart-level trial). Muscle sympathetic nerve activity (MSNA), blood pressure and heart rate were monitored. MSNA increased during handgrip exercise in both forearm positions (P<0.001); the increase was 51% greater in the forearm-elevated trial (516+/-99 arbitrary units) than in the heart-level trial (346+/-44 units; P<0.05). The increase in mean blood pressure was 8.4 mmHg greater during exercise in the forearm-elevated trial (P<0.05), while changes in heart rate were similar in both forearm positions. The increase in MSNA during PEMI was 71% greater in the forearm-elevated trial (393+/-71 arbitrary units/min) than in the heart-level trial (229+/-29 units/min; P<0.05). These results support the hypothesis that forearm elevation augments sympathetic activation during handgrip exercise. The excitatory effect of forearm elevation on exercising MSNA may be mediated primarily by increased activation of the muscle metaboreflex.