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Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurological impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remain unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2-/- ) neurons exhibited elevated neuronal activity with highly synchronized Ca2+ spikes. Notably, TSC2-/- neurons presented enhanced Ca2+ influx via L-type Ca2+ channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2-/- neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2-/- neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC.Significant statementThere is a close relationship between elevated mTOR activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2-/- neurons, we identified elevated Ca2+ influx via LTCCs as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2-/- neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca2+ signaling via LTCC in human TSC neurons, which could cause epilepsy in TSC.

Tuberous sclerosis complex (TSC) is caused by mutations inTsc1orTsc2, whose gene products inhibit the small G-protein Rheb1. Rheb1 activates mTORC1, which may cause refractory epilepsy, intellectual disability, and autism. The mTORC1 inhibitors have been used for TSC patients with intractable epilepsy. However, its effectiveness for cognitive symptoms remains unclear. We found a new signaling pathway for synapse formation through Rheb1 activation, but not mTORC1. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib increased unfarnesylated (inactive) Rheb1 levels and restored synaptic abnormalities in culturedTsc2+/−neurons, whereas rapamycin did not enhance spine synapse formation. Lonafarnib treatment also restored the plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in culturedTsc2+/−neurons. Lonafarnib action was partly dependent on the Rheb1 reduction with syntenin. Oral administration of lonafarnib increased unfarnesylated protein levels without affecting mTORC1 and MAP (mitogen-activated protein (MAP)) kinase signaling, and restored dendritic spine morphology in the hippocampi of maleTsc2+/−mice. In addition, lonafarnib treatment ameliorated contextual memory impairments and restored memory-related Arc expression in maleTsc2+/−micein vivo. HeterozygousRheb1knockout in maleTsc2+/−mice reproduced the results observed with pharmacological treatment. These results suggest that the Rheb1 activation may be responsible for synaptic abnormalities and memory impairments inTsc2+/−mice, and its inhibition by lonafarnib could provide insight into potential treatment options for TSC-associated neuropsychiatric disorders.SIGNIFICANCE STATEMENTTuberous sclerosis complex (TSC) is an autosomal-dominant disease that causes neuropsychiatric symptoms, including intractable epilepsy, intellectual disability (ID) and autism. No pharmacological treatment for ID has been reported so far. To develop a pharmacological treatment for ID, we investigated the mechanism of TSC and found that Rheb1 activation is responsible for synaptic abnormalities in TSC neurons. To inhibit Rheb1 function, we used the farnesyltransferase inhibitor lonafarnib, because farnesylation of Rheb1 is required for its activation. Lonafarnib treatment increased inactive Rheb1 and recovered proper synapse formation and plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in TSC neurons. Furthermore,in vivolonafarnib treatment restored contextual memory and Arc induction in TSC mice. Together, Rheb1 inhibition by lonafarnib could provide insight into potential treatments for TSC-associated ID.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs, such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurologic impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remains unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2(−/−)) neurons exhibited elevated neuronal activity with highly synchronized Ca(2+) spikes. Notably, TSC2(−/−) neurons presented enhanced Ca(2+) influx via L-type Ca(2+) channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2(−/−) neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2(−/−) neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC. SIGNIFICANCE STATEMENT There is a close relationship between elevated mammalian target of rapamycin (mTOR) activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2(−/−) neurons, we identified elevated Ca(2+) influx via L-type Ca(2+) channels as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2(−/−) neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca(2+) signaling via L-type Ca(2+) channels in human TSC neurons, which could cause epilepsy in TSC.

Neuritin is a small extracellular protein that plays important roles in the process of neural development, synaptic plasticity, and neural cell survival. Here we investigated the function of neuritin in a mouse model of optic nerve injury (ONI). ONI induced upregulation of neuritin mRNA in the retina of WT mice. The retinal structure and the number of retinal ganglion cells (RGCs) were normal in adult neuritin knockout (KO) mice. In vivo retinal imaging and histopathological analyses demonstrated that RGC death and inner retinal degeneration following ONI were more severe in neuritin KO mice. Immunoblot analyses revealed that ONI-induced phosphorylation of Akt and ERK were suppressed in neuritin KO mice. Our findings suggest that neuritin has neuroprotective effects following ONI and may be useful for treatment of posttraumatic complication.

Aberrant branch formation of granule cell axons (mossy fiber sprouting) is observed in the dentate gyrus of many patients with temporal lobe epilepsy and in animal models of epilepsy. However, the mechanisms underlying mossy fiber sprouting remain elusive. Based on the hypothesis that seizure-mediated gene expression induces abnormal mossy fiber growth, we screened activity-regulated genes in the hippocampus and found that neuritin, an extracellular protein anchored to the cell surface, was rapidly upregulated after electroconvulsive seizures. Overexpression of neuritin in the cultured rat granule cells promoted their axonal branching. Also, kainic acid-dependent axonal branching was abolished in the cultured granule cells fromneuritinknock-out mice, suggesting that neuritin may be involved in activity-dependent axonal branching. Moreover,neuritinknock-out mice showed less-severe seizures in chemical kindling probably by reduced mossy fiber sprouting and/or increased seizure resistance. We found that inhibition of the fibroblast growth factor (FGF) receptor attenuated the neuritin-dependent axonal branching. FGF administration also increased branching in granule neurons, whereasneuritinknock-out mice did not show FGF-dependent axonal branching. In addition, FGF and neuritin treatment enhanced the recruitment of FGF receptors to the cell surface. These findings suggest that neuritin and FGF cooperate in inducing mossy fiber sprouting through FGF signaling. Together, these results suggest that FGF and neuritin-mediated axonal branch induction are involved in the aggravation of epilepsy.SIGNIFICANCE STATEMENTThis study reveals the molecular mechanism underlying mossy fiber sprouting. Mossy fiber sprouting is the aberrant axonal branching of granule neurons in the hippocampus, which is observed in patients with epilepsy. Excess amounts of neuritin, a protein upregulated by neural activity, promoted axonal branching in granule neurons. A deficiency of neuritin suppressed mossy fiber sprouting and resulted in mitigation of seizure severity. Neuritin and fibroblast growth factor (FGF) cooperated in stimulating FGF signaling and enhancing axonal branching. Neuritin is necessary for FGF-mediated recruitment of FGF receptors to the cell surface. The recruitment of FGF receptors would promote axonal branching. The discovery of this new mechanism should contribute to the development of novel antiepileptic drugs to inhibit axonal branching via neuritin-FGF signaling.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.

Syntenin is an adaptor-like molecule that has two adjacent tandem postsynaptic density protein 95/Discs large protein/Zonula occludens 1 (PDZ) domains. The PDZ domains of syntenin recognize multiple peptide motifs with low to moderate affinity. Many reports have indicated interactions between syntenin and a plethora of proteins. Through interactions with various proteins, syntenin regulates the architecture of the cell membrane. As a result, increases in syntenin levels induce the metastasis of tumor cells, protrusion along the neurite in neuronal cells, and exosome biogenesis in various cell types. Here, we review the updated data that support various roles for syntenin in the regulation of neuronal synapses, tumor cell invasion, and exosome control.

Growth cones regulate the speed and direction of neuronal outgrowth during development and regeneration. How the growth cone spatially and temporally regulates signals from guidance cues is poorly understood. Through a proteomic analysis of purified growth cones we identified isoforms of the 14-3-3 family of adaptor proteins as major constituents of the growth cone. Disruption of 14-3-3 via the R18 antagonist or knockdown of individual 14-3-3 isoforms switches nerve growth factor- and myelin-associated glycoprotein-dependent repulsion to attraction in embryonic day 13 chick and postnatal day 5 rat DRG neurons. These effects are reminiscent of switching responses observed in response to elevated cAMP. Intriguingly, R18-dependent switching is blocked by inhibitors of protein kinase A (PKA), suggesting that 14-3-3 proteins regulate PKA. Consistently, 14-3-3 proteins interact with PKA and R18 activates PKA by dissociating its regulatory and catalytic subunits. Thus, 14-3-3 heterodimers regulate the PKA holoenzyme and this activity plays a critical role in modulating neuronal responses to repellent cues.

Actin polymerizes near the leading edge of nerve growth cones, and actin filaments show retrograde movement in filopodia and lamellipodia. Linkage between actin filament retrograde flow and cell adhesion molecules (CAMs) in growth cones is thought to be one of the mechanisms for axon outgrowth and guidance. However, the molecular basis for this linkage remains elusive. Here, we show that shootin1 interacts with both actin filament retrograde flow and L1-CAM in axonal growth cones of cultured rat hippocampal neurons, thereby mediating the linkage between them. Impairing this linkage, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, inhibited L1-dependent axon outgrowth, whereas enhancing the linkage by shootin1 overexpression promoted neurite outgrowth. These results strengthen the actin flow–CAM linkage model (“clutch” model) for axon outgrowth and suggest that shootin1 is a key molecule involved in this mechanism.

Neurons have the remarkable ability to polarize even in symmetrical in vitro environments. Although recent studies have shown that asymmetric intracellular signals can induce neuronal polarization, it remains unclear how these polarized signals are organized without asymmetric cues. We describe a novel protein, named shootin1, that became up-regulated during polarization of hippocampal neurons and began fluctuating accumulation among multiple neurites. Eventually, shootin1 accumulated asymmetrically in a single neurite, which led to axon induction for polarization. Disturbing the asymmetric organization of shootin1 by excess shootin1 disrupted polarization, whereas repressing shootin1 expression inhibited polarization. Overexpression and RNA interference data suggest that shootin1 is required for spatially localized phosphoinositide-3-kinase activity. Shootin1 was transported anterogradely to the growth cones and diffused back to the soma; inhibiting this transport prevented its asymmetric accumulation in neurons. We propose that shootin1 is involved in the generation of internal asymmetric signals required for neuronal polarization.

Mutations in the Tsc1 or Tsc2 genes cause tuberous sclerosis complex (TSC). Tsc1 and Tsc2 proteins form a complex that inhibits mammalian target of rapamycin complex 1 (mTORC1) signalling through Rheb-GTPase. We found that Tsc2(+/-) neurons showed impaired spine synapse formation, which was resistant to an mTORC1 inhibitor. Knockdown of mTOR also failed to restore these abnormalities, suggesting mTORC may not participate in impaired spinogenesis in Tsc2(+/-) neurons. To address whether Rheb activation impairs spine synapse formation, we expressed active and inactive forms of Rheb in WT and Tsc2(+/-) neurons, respectively. Expression of active Rheb abolished dendritic spine formation in WT neurons, whereas inactive Rheb restored spine synapse formation in Tsc2(+/-) neurons. Moreover, inactivation of Rheb with farnesyl transferase inhibitors recovered spine synapse morphogenesis in Tsc2(+/-) neurons. In conclusion, dendritic spine abnormalities in TSC neurons may be caused through activation of Rheb, but not through of mTORC1.

Epilepsy is one of the most common chronic brain disorders worldwide, affecting 1% of people across different ages and backgrounds. Epilepsy is defined as the sporadic occurrence of spontaneous recurrent seizures. Accumulating preclinical and clinical evidence suggest that there is a positive feedback cycle between epileptogenesis and brain inflammation. Epileptic seizures increase key inflammatory mediators, which in turn cause secondary damage to the brain and increase the likelihood of recurrent seizures. Cytokines and prostaglandins are well-known inflammatory mediators in the brain, and their biosynthesis is enhanced following seizures. Such inflammatory mediators could be therapeutic targets for the development of new antiepileptic drugs. In this review, we discuss the roles of inflammatory mediators in epileptogenesis.

14-3-3 proteins are abundantly expressed adaptor proteins that interact with a vast number of binding partners to regulate their cellular localization and function. They regulate substrate function in a number of ways including protection from dephosphorylation, regulation of enzyme activity, formation of ternary complexes and sequestration. The diversity of 14-3-3 interacting partners thus enables 14-3-3 proteins to impact a wide variety of cellular and physiological processes. 14-3-3 proteins are broadly expressed in the brain, and clinical and experimental studies have implicated 14-3-3 proteins in neurodegenerative disease. A recurring theme is that 14-3-3 proteins play important roles in pathogenesis through regulating the subcellular localization of target proteins. Here, we review the evidence that 14-3-3 proteins regulate aspects of neurodegenerative disease with a focus on their protective roles against neurodegeneration.

本論文は, ケーブルの高次モードの固有振動数から張力を測定することを目的として, ケーブルのサグ・傾斜・曲げ剛性を考慮したケーブル振動の特性方程式を導き, 測定精度についての考察を行った. この結果, 高次振動次数から推定した張力は十分なる精度を有することが判明し, この方法を用いれば常時微動法が適用できるので, 長さの長いケーブルに対しても加振する必要がなくなり, 迅速な測定ができることを示した.

Shootin1, one of the earliest markers of neuronal symmetry breaking, accumulates in the neurite tips of polarizing neurons in a neurite length-dependent manner. Thus, neurons sense their neurites' length and translate this spatial information into a molecular signal, shootin1 concentration. Quantitative live cell imaging of shootin1 dynamics combined with mathematical modeling analyses reveals that its anterograde transport and retrograde diffusion in neurite shafts account for the neurite length-dependent accumulation of shootin1. The neurite length-dependent shootin1 accumulation and shootin1-induced neurite outgrowth constitute a positive feedback loop that amplifies stochastic shootin1 signals in neurite tips. Quantitative mathematical modeling shows that the above positive feedback loop, together with shootin1 upregulation, constitutes a core mechanism for neuronal symmetry breaking., Cell morphology and size must be properly controlled to ensure cellular function. Although there has been significant progress in understanding the molecular signals that change cell morphology, the manner in which cells monitor their size and length to regulate their morphology is poorly understood. Cultured hippocampal neurons polarize by forming a single long axon and multiple short dendrites (Craig and Banker, 1994; Arimura and Kaibuchi, 2007), and symmetry breaking is the initial step of this process. This symmetry-breaking step reproduces even when the neuronal axon is transected; the longest neurite usually grows rapidly to become an axon after transection, regardless of whether it is the axonal stump or another neurite (Goslin and Banker, 1989). Elongation of an immature neurite by mechanical tension also leads to its axonal specification (Lamoureux et al, 2002). These results suggest that cultured hippocampal neurons can sense neurite length, identify the longest one, and induce its subsequent axonogenesis for symmetry breaking. However, little is known about the mechanism for this process. Shootin1 is one of the earliest markers of neuronal symmetry breaking (Toriyama et al, 2006). During the symmetry-breaking step, it undergoes a stochastic accumulation in neurite tips, and eventually accumulates predominantly in a single neurite that subsequently grows to become an axon. In this study, we demonstrated that shootin1 accumulates in neurite tips in a neurite length-dependent manner, regardless of whether it is the axonal stump or another neurite (Figure 3A, C–F). Thus, morphological information (neurite length) is translated into a molecular signal (shootin1 concentration in neurite tips). We previously reported that shootin1 is transported from the cell body to neurite tips as discrete boluses and diffuses back to the cell body (Toriyama et al, 2006). The boluses containing variable amounts of shootin1 traveled repeatedly but irregularly along neurites, and their arrival caused large stochastic fluctuations in shootin1 concentration in the neurite tips. To understand the mechanism of length-dependent shootin1 accumulation, we performed quantitative live cell imaging of the anterograde transport and retrograde diffusion of shootin1 and fitted the obtained data into mathematical models of the anterograde transport and retrograde diffusion. The parameters of these two models were derived entirely from quantitative experimental data, without any adjustment. Shootin1 concentration at neurite tips, calculated by integrating the two models, was neurite length dependent (Figure 3B) and showed good agreement with the experimental data (Figure 3A). These results suggest that the neurite length-dependent accumulation of shootin1 is quantitatively explained by its anterograde transport and retrograde diffusion. This length-dependent shootin1 accumulation constitutes a positive feedback interaction with the previously reported shootin1-induced neurite outgrowth (Shimada et al, 2008). To analyze the functional role of this feedback loop, we quantified shootin1 upregulation (Toriyama et al, 2006) and shootin1-induced neurite outgrowth, and integrated them, together with the above model of length-dependent shootin1 accumulation, into a model neuron (Figure 7A). Furthermore, the parameters of the model components were chosen to give the best fit to the quantitative experimental data without any adjustment. Integrating the three components into a model neuron resulted in spontaneous symmetry breaking (Figure 7B and C). Furthermore, there are a total of 15 agreements between the model predictions and the experimental data, including the neurite length-dependent axon specification and regeneration (Goslin and Banker, 1989; Lamoureux et al, 2002). These data suggest that the three components in our model—namely, diffusion-based neurite length sensing system, shootin1-induced neurite outgrowth and shootin1 upregulation—are sufficient to induce neuronal symmetry breaking. Bolus-like transport of shootin1 caused large stochastic fluctuations in shootin1 concentration in neurite tips. Interestingly, the generation of continuous shootin1 transport in our model neuron impaired the symmetry-breaking process (Figure 7D). This is consistent with theoretical models in which feedback amplification of fluctuations in signaling can give rise to robust patterns (Turing, 1952; Meinhardt and Gierer, 2000; Kondo, 2002), and underscores the importance of the stochastic fluctuating signals in spontaneous neuronal symmetry breaking. The combination of quantitative experimentation and mathematical modeling is regarded as a powerful strategy for attaining a profound understanding of biological systems (Hodgkin and Huxley, 1952b; Lewis, 2008; Ferrell, 2009). By focusing on a simple system involving one of the earliest markers of neuronal symmetry breaking, shootin1, we were able to evaluate here the core components of neuronal symmetry breaking on the basis of quantitative experimental data. The present model may thus provide a core mechanism of neuronal symmetry breaking, to which other possible mechanisms can be added to increase the model's complexity in future studies., Although there has been significant progress in understanding the molecular signals that change cell morphology, mechanisms that cells use to monitor their size and length to regulate their morphology remain elusive. Previous studies suggest that polarizing cultured hippocampal neurons can sense neurite length, identify the longest neurite, and induce its subsequent outgrowth for axonogenesis. We observed that shootin1, a key regulator of axon outgrowth and neuronal polarization, accumulates in neurite tips in a neurite length-dependent manner; here, the property of cell length is translated into shootin1 signals. Quantitative live cell imaging combined with modeling analyses revealed that intraneuritic anterograde transport and retrograde diffusion of shootin1 account for its neurite length-dependent accumulation. Our quantitative model further explains that the length-dependent shootin1 accumulation, together with shootin1-dependent neurite outgrowth, constitutes a positive feedback loop that amplifies stochastic fluctuations of shootin1 signals, thereby generating an asymmetric signal for axon specification and neuronal symmetry breaking.

The method of estimating tension of bridge cables by measuring their natural frequency, so-called vibration method, is convenient on construction site of structures with cables, Concerning the method some papers have been reported hitherto. In these reports cables are assumed to be clamped on both ends. However, for the estimated tension of cable supported by open sockets, vibration of the sockets has a notable effect, which has not been investigated. In order to get reliable and accurate estimation for tension of cables with open socket, some experimental works were performed and reported in this paper.

Fission yeast Mei2p is an RNA-binding protein essential for induction of both premeiotic DNA synthesis and first meiotic division. Mei2p forms a dot structure at an apparently fixed position in the horse-tail nucleus during meiotic prophase. This dot formation requires a meiosis-specific RNA species, meiRNA, which is indispensable for meiosis I, and the emergence of the dot is an indicator of the ability of the cell to perform meiosis I. Herein, we have sought the identity of this dot. Analyses using chromosome segregation in haploid meiosis, reciprocal translocation of chromosomes, and gene translocation have led us to conclude that the Mei2p dot is in association with the sme2 gene on the short arm of chromosome II, which encodes meiRNA. Transcripts of sme2, rather than the DNA sequence of the gene, seem to be the determinant of the localization of the Mei2p dot. However, evidence suggests that the dot may not be a simple reflection of the attachment of Mei2p to meiRNA undergoing transcription. We speculate that the Mei2p dot is a specialized structure, either to foster the assembly of Mei2p and meiRNA or to perform some unidentified function indispensable for meiosis I.

The authors have been researching the promotion of lactic acid fermentation by ultrasonic irradiation. In the past research, it was proven that ultrasonic irradiation is effective in the process of fermentation, and the production of yoghurt and kefir was promoted. In this study, the effect of the ultrasonic frequency in this fermentation process was examined. In the frequency range of this study, it was found that the action of fermentation promotion was exponentially proportionate to the irradiated ultrasonic frequency.

The method of estimating tension of bridge cables by measuring their natural frequency, so-called vibration method, is a very convenient way at the construction site of structures with cables. However in order to apply the method to estimate hanger-rope tension of a suspension bridge which has long spans, there still exist several problems to be solved as shown below because those ropes are tied with hanger clamps.1) The vibration mode of two hanger ropes connected with a hanger clamp is needed to research beforehand.2) The effect of hanger-clamp weight on estimated tension is necessary to investigate beforehand.3) The accuracy of tension estimated by vibration method is necessary to compare with tension measured by a loadcell so as to get reliable estimation.In this paper, the above mentioned problems are discussed and checked.

Recently the use of wind energy is becoming the center of public interest in.Japan and in1981the project of developing100KW class wind turbine generator started.Io design the wind turbine generator, it is necessary to solve many technical problems such as performance, control, vibration and structural strength, but these problems are often clarified by wind obServation at the site.For this purpose the wind observation has beer conducted ai Miyake Island smoc1980, which had been selected at the construction place.This paper describes about the site measurement and investigation on the design based on it.

Helical strikes are well known as a device to suppress the vortex-excited oscillation of stacks and have been applied to some stacks to prevent the oscillation. Wind tunnel experiments on two-dimensional models were performed to reconsider the optimum configuration of the strakes. Based on the model experimental results, the effectiveness of the strakes was confirmed by experiments on an actual stack. Finally, the stack with strakes was shown to be more economical than the plain stack without strakes.