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Purpose：Sleep apnea syndrome (SAS) is a prevalent sleep disorder in which apnea and hypopnea occur frequently during sleep and result in increase of the risk of lifestyle-related disease development as well as daytime sleepiness. Although SAS is a common sleep disorder, most patients remain undiagnosed because the gold standard test polysomnography (PSG), is high-cost and unavailable in many hospitals. Thus, an SAS screening system that can be used easily at home is needed., Methods：Apnea during sleep affects changes in the autonomic nervous function, which causes fluctuation of the heart rate. In this study, we propose a new SAS screening method that combines heart rate measurement and long short-term memory (LSTM) which is a type of recurrent neural network (RNN). We analyzed the data of intervals between adjacent R waves (R-R interval; RRI) on the electrocardiogram (ECG) records, and used an LSTM model whose inputs are the RRI data is trained to discriminate the respiratory condition during sleep., Results：The application of the proposed method to clinical data showed that it distinguished between patients with moderate-to-severe SAS with a sensitivity of 100% and specificity of 100%, results which are superior to any other existing SAS screening methods., Conclusion：Since the RRI data can be easily measured by means of wearable heart rate sensors, our method may prove to be useful as an SAS screening system at home.

Objective:Easily detecting patients with undiagnosed sleep apnea syndrome (SAS) requires a home-use SAS screening system. In this study, we validate a previously developed SAS screening methodology using a large clinical polysomnography (PSG) dataset (N = 938)., Methods:We combined R-R interval (RRI) and long short-term memory (LSTM), a type of recurrent neural networks, and created a model to discriminate respiratory conditions using the training dataset (N = 468). Its performance was validated using the validation dataset (N = 470)., Results:Our method screened patients with severe SAS (apnea hypopnea index; AHI ≥ 30) with an area under the curve (AUC) of 0.92, a sensitivity of 0.80, and a specificity of 0.84. In addition, the model screened patients with moderate/severe SAS (AHI ≥ 15) with an AUC of 0.89, a sensitivity of 0.75, and a specificity of 0.87., Conclusions:Our method achieved high screening performance when applied to a large clinical dataset., Significance:Our method can help realize an easy-to-use SAS screening system because RRI data can be easily measured with a wearable heart rate sensor. It has been validated on a large dataset including subjects with various backgrounds and is expected to perform well in real-world clinical practice.

Background:Orthostatic hypotension (OH) caused by autonomic dysfunction is a common symptom in older people and patients with idiopathic rapid eye movement sleep behavior disorder (iRBD). The orthostatic challenge test is a standard autonomic function test that measures a decrease of blood pressure during a postural change from supine to standing positions. Although previous studies have reported that changes in heart rate variability (HRV) are associated with autonomic dysfunction, no study has investigated the relationship between HRV before standing and the occurrence of OH in an orthostatic challenge test. This study aims to examine the connection between HRV in the supine position and the occurrence of OH in an orthostatic challenge test., Methods:We measured the electrocardiograms of patients with iRBD and healthy older people during an orthostatic challenge test, in which the supine and standing positions were held for 15 min, respectively. The subjects were divided into three groups: healthy controls (HC), OH-negative iRBD [OH (–) iRBD], and OH-positive iRBD [OH (+) iRBD]. HRV measured in the supine position during the test were calculated by time-domain analysis and Poincaré plots to evaluate the autonomic dysfunction., Results:Forty-two HC, 12 OH (–) iRBD, and nine OH (+) iRBD subjects were included. HRV indices in the OH (–) and the OH (+) iRBD groups were significantly smaller than those in the HC group. The multivariate logistic regression analysis for OH identification for the iRBD groups showed the model whose inputs were the HRV indices, i.e., standard deviation 2 (SD2) and the percentage of adjacent intervals that varied by more than 50 ms (pNN50), had a receiver operating characteristic curve with area under the curve of 0.840, the sensitivity to OH (+) of 1.000, and the specificity to OH (–) of 0.583 (p = 0.023)., Conclusions:This study showed the possibility that short-term HRV indices in the supine position would predict subsequent OH in iRBD patients. Our results are of clinical importance in terms of showing the possibility that OH can be predicted using only HRV in the supine position without an orthostatic challenge test, which would improve the efficiency and safety of testing.

Sleep apnea syndrome (SAS) is a prevalent disorder which causes daytime fatigue with the increased risk of lifestyle diseases. A large number of patients are undiagnosed and untreated partly because of the difficulty in performing its gold standard test, polysomnography (PSG). In this research, we propose a simple screening method utilizing heart rate variability (HRV) and long short-term memory (LSTM) which is a kind of neural network techniques. The result of applying this algorithm to clinical data demonstrates that it can discriminate between patients and healthy people with high sensitivity (100%) and specificity (100%).

Objective: Obstructive sleep apnea (OSA) is a common sleep disorder; however, most patients are undiagnosed and untreated because it is difficult for patients themselves to notice OSA in daily living. Polysomnography (PSG), which is the gold standard test for sleep disorder diagnosis, cannot be performed in many hospitals. This fact motivates us to develop a simple system for screening OSA at home. Approach: The autonomic nervous system changes during apnea, and such changes affect heart rate variability (HRV). This work develops a new apnea screening method based on HRV analysis and machine learning technologies. An apnea/normal respiration (A/N) discriminant model is built for respiration condition estimation for every heart rate measurement, and an apnea/sleep ratio is introduced for final diagnosis. A random forest is adopted for the A/N discriminant model construction, which is trained with the PhysioNet apnea-ECG database. Main results: The screening performance of the proposed method was evaluated by applying it to clinical PSG data. Sensitivity and specificity achieved 76% and 92%, respectively, which are comparable to existing portable sleep monitoring devices used in sleep laboratories. Significance: Since the proposed OSA screening method can be used more easily than existing devices, it will contribute to OSA treatment.

Objective: Driver drowsiness detection is a key technology that can prevent fatal car accidents caused by drowsy driving. The present work proposes a driver drowsiness detection algorithm based on heart rate variability (HRV) analysis and validates the proposed method by comparing with electroencephalography (EEG)-based sleep scoring. Methods: Changes in sleep condition affect the autonomic nervous system and then HRV, which is defined as an RR interval (RRI) fluctuation on an electrocardiogram trace. Eight HRV features are monitored for detecting changes in HRV by using multivariate statistical process control, which is a well known anomaly detection method. Result: The performance of the proposed algorithm was evaluated through an experiment using a driving simulator. In this experiment, RRI data were measured from 34 participants during driving, and their sleep onsets were determined based on the EEG data by a sleep specialist. The validation result of the experimental data with the EEG data showed that drowsiness was detected in 12 out of 13 pre-N1 episodes prior to the sleep onsets, and the false positive rate was 1.7 times per hour. Conclusion: The present work also demonstrates the usefulness of the framework of HRV-based anomaly detection that was originally proposed for epileptic seizure prediction. Significance: The proposed method can contribute to preventing accidents caused by drowsy driving.

Objective Hemifacial spasm (HFS) is caused by arterial conflict at the root exit zone of the facial nerve. As the offending artery is pulsatile in nature, this study investigated the association of heart rate fluctuation with HFS. Methods Twenty-four preoperative patients underwent simultaneous recordings of facial electromyogram and electrocardiogram overnight. Series of R-wave to R-wave intervals (RRIs) in the electrocardiogram were analyzed across subjects in relation to HFS. The degree of heart rate fluctuation was quantified by analyzing the heart rate variability (HRV). The sleep stage was evaluated during the period of HFS. Results A 0.1 Hz fluctuation in RRIs by 5% compared to the baseline preceded a few seconds the onset of the HFS, indicating that a significant increase in the heart rate coincided with HFS. HRV analysis demonstrated that fluctuations in the heart rate were significantly enhanced during HFS. Wake or light sleep stages were more often accompanied by HFS, suggesting an association with autonomic activities. Conclusion Our findings suggest that the etiology of HFS is more than just a mechanical compression of the facial nerve and may involve changes in pulsatile frequency in offending arteries. Significance We propose the etiology of HFS from a unique standpoint.

Although sleep apnea syndrome (SAS) is a common sleep disorder, most patients with sleep apnea are undiagnosed and untreated because it is difficult for patients themselves to notice SAS in daily living. Polysomnography (PSG) is a gold standard test for sleep disorder diagnosis, however PSG cannot be performed in many hospitals. This fact motivates us to develop an SAS screening system that can be used easily at home. The autonomic nervous function of a patient changes during apnea. Since changes in the autonomic nervous function affect fluctuation of the R-R interval (RRI) of an electrocardiogram (ECG), called heart rate variability (HRV), SAS can be detected through monitoring HRV. The present work proposes a new HRV-based SAS screening algorithm by utilizing support vector machine (SVM), which is a well-known pattern recognition method. In the proposed algorithm, various HRV features are derived from RRI data in both apnea and normal respiration periods of patients and healthy people, and an apnea/normal respiration (A/N) discriminant model is built from the derived HRV features by SVM. The result of applying the proposed SAS screening algorithm to clinical data demonstrates that it can discriminate patients with sleep apnea and healthy people appropriately. The sensitivity and the specificity of the proposed algorithm were 100% and 86%, respectively.

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

We synthesized lecithinized brain-derived neurotrophic factor (lecithinized-BDNF), in which an average of three molecules of a lecithin derivative were bound to recombinant human BDNF. We evaluated its pharmacological activity in C57BL/KsJ-db/db mice, and assessed its targetability and affinity for the nervous system. Subcutaneously administered lecithinized-BDNF markedly reduced the plasma glucose level, food intake, and body weight in C57BL/KsJ-db/db diabetic mice. Its potency was more than 20 times greater than that of unmodified BDNF. We then studied the mechanism for the markedly enhanced pharmacological activity. In vitro cell growth activity of lecithinized-BDNF using the MTT assay was lower than unmodified BDNF, probably due to steric hindrance of the lecithin moieties. While the plasma BDNF level after subcutaneous administration of lecithinized-BDNF was not higher compared with unmodified BDNF. However, higher amount of lecithinized-BDNF accumulated in the spinal cord was observed. Lastly, we found that in vitro binding capacity of lecithinized-BDNF for PC-pAB1 neural cells was much higher than unmodified BDNF. Moreover, lecithinized-BDNF bound to PC-pAB1 cells did not exchange with an excessive amount of unmodified BDNF or an excess of lecithinized-BDNF. PC-pAB1 cells treated with lecithinized-BDNF showed sustained mitogen-activated protein kinase (MAPK, ERK1/2) activation. These data would indicate that the high affinity of lecithinized-BDNF for the target cells, followed by prolonged MAPK activation, would play an important role in its potent pharmacological activity.

In the wobbler mouse motor neuron disease (MND), we firstly evaluated the effect of riluzole, the only approved drug for amyotrophic lateral sclerosis, and compared it with that of brain-derived neurotrophic factor (BDNF). Wobbler mice received either daily subcutaneous treatment with BDNF (5, 20, and 40 mg/kg) or oral riluzole in drinking water (100 and 200 microg/ml), beginning immediately after the clinical onset of MND. We examined motor functions, such as grip strength and rota-rod walking performance, weekly, and the amplitude of the compound muscle action potential (CMAP) in the forelimb biceps at the end of treatment. BDNF treatment slowed the disease progression maximally at a dose of 20 mg/kg, consistent to the previous evidence. Only high-dose riluzole treatment increased grip strength at weeks 1 (P=0.0023) and 2 (P=0.021), time before falling in the rota-rod test throughout all 4 weeks of treatment (P=0.0022 to 0.0282), and CMAP amplitude (P=0.0069) at the end of treatment, compared with the vehicle. Furthermore, the riluzole treatment increased the number of the cervical cord anterior horn neurons that were immunoreactive for SMI-32, a specific motor neuron marker, by the end of treatment (P=0.0063), although it did not affect the vacuolar degeneration on the SMI-32-positive neurons. This study demonstrated that riluzole was comparable to BDNF in slowing the progression of neuromuscular dysfunction in the wobbler mouse MND, which may provide a useful model for examining the mechanisms of selective motor neuron degeneration.

Neurotrophic factors and neurotrophins are well-known to have neuroprotective efficacy against retinal injury. The aim of this experiment is to investigate the signal transduction pathway of ciliary neurotrophic factor (CNTF) on the upregulation of viability of retinal primary culture and retinal protection against constant light damage in vivo. CNTF is known to enhance the viability of retinal culture and provide protection under constant light exposure conditions, but little is known about how the signal transduction pathways of CNTF affect retina function.Primary retinal cultures were prepared from 7-day-old Wistar rats. Brain-derived neurotrophic factor (BDNF) (0.1, 1, 10 ng/ml), CNTF (0.1, 1, 10 ng/ml), PD98059 (10, 100, 1000 nM), or LY294002 (10, 100, 1000 nM) was added to these cultures at the time of cell preparation. After 3 days, the percentage of cells surviving was assessed using alamarBlue. For the in vivo experiment, inhibitors for the MAPKK (PD98059, 10 microg/eye) or PI3K (LY294002, 10 microg/eye) pathways were injected into the vitreous together with CNTF (1 microg/eye) 2 days before constant light exposure. Electroretinogram (ERG) analysis was performed to investigate which pathway was used by CNTF.CNTF at 1, 10, or 100 ng/ml enhanced cell viability in retinal cultures. The cell-survival activity of CNTF was blocked by 10 ng/ml LY294002 (Dunnet's test, p0.05). In vivo, the neuroprotective activity of CNTF in constant-light conditions was attenuated by 10 microg/eye LY294002 (Dunnet's test, p0.05).These data suggest that CNTF promotes cell survival via the PI3K signaling pathway in vitro and in vivo.

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 mum (IC50 = 0.16 mum). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries.

We investigated the neuroprotective effects of brain-derived neurotrophic factor (BDNF) and its influence on the functional recovery of the retina following light-induced retinal damage by electroretinogram (ERG). Rats were exposed to constant fluorescent light for 2, 5, 7, or 14 days, then returned to a cyclic light environment for 14 days. The result indicated that BDNF had few effects on the a-wave amplitude, but there was a statistically significant difference in the b-wave amplitudes between BDNF-treated and control eyes from day 0-14 of the recovery period following 2 days of light exposure (p < 0.05). Our findings suggest that BDNF not only protects the retinal neuronal function but also enhances the recovery from retinal light damage.

The structure-function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival-promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival-promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.

We compared the clinical and pharmacodynamic effects of N-terminal methionine brain-derived neurotrophic factor (met-BDNF) and endogenous met-free BDNF in wobbler mouse motor neuron disease (MND). Met- or met-free BDNF at 5 or 20 mg/kg was subcutaneously injected daily, six times/week for 4 weeks. At 20 mg/kg, grip strength (P

We evaluated the effect of brain-derived neurotrophic factor (BDNF) on cell death after spinal cord injury. A rat spinal cord injury model was produced by static load, and continuous intrathecal BDNF or vehicle infusion was carried out either immediately or 3 days after the injury. Cell death was examined by nuclear staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After injury, typical apoptotic cells were observed. Double staining with TUNEL and specific cell markers revealed that, soon after the injury, the apoptotic or necrotic cells at the injury site were neurons and microglia. One week after the injury, apoptotic oligodendrocytes, but not apoptotic astrocytes, were observed in the white matter rostral and caudal to the injury site, whereas few apoptotic cells were found in the gray matter. The immediate BDNF treatment significantly reduced the number of TUNEL-positive cells in the adjacent rostral site 1 and 2 weeks after the injury, and in the adjacent caudal site 3 days and 1 week after the injury, even though there was no significant difference between BDNF-treated and control rats at the injury site itself. In addition, similar antiapoptotic effects were observed in these regions 1 week after injury in rats that received BDNF treatment from the third day after injury. These findings suggest that BDNF suppresses delayed apoptosis of oligodendrocytes after spinal cord injury, for which even delayed injections are effective. BDNF administration may therefore be useful for the clinical treatment of spinal cord injury through the suppression of secondary events.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family of trophic factors, has multiple functions including a role in the promotion of neuronal survival and nerve fiber elongation in both the central and the peripheral nervous systems. We assessed the expression of endogenous BDNF following an experimentally induced compression injury to the spinal cord. Expression of BDNF mRNA was increased following the spinal cord injury; reaching maximum levels 24 h after the injury. Expression of BDNF mRNA returned to the levels observed in sham-operated control animals within 3 days of the injury. Using the in situ hybridization technique, we observed a wide distribution of BDNF expression among the different cell types in the spinal cord, including motor and sensory neurons, and in glia cells, including astrocytes. We also observed expression of BDNF in putative macrophages and/or microglia; however, this effect was not observed until day 7 following spinal cord injury. These results suggest that BDNF is synthesized in both neurons and astrocytes during the acute response to injury to the spinal cord, functioning in a mainly neuroprotective role. This is followed by a later phase of expression in which BDNF is produced by macrophages and/or microglia, apparently functioning in a restorative capacity.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. However, it is not yet known if BDNF works on the endocrine system itself. Here we report that BDNF improves hyperglycemia in obese diabetic animals. BDNF reduced the blood glucose level in obese db/db diabetic mice in which the effect of BDNF was age-dependent and high under the condition of hyperinsulinemia, while BDNF showed no effect on non-diabetic db/m mice. These results suggest that BDNF ameliorates insulin resistance by enhancing insulin action in peripheral tissues. Furthermore, BDNF was found to reduce the plasma insulin level in db/db mice. Among the neurotrophin family, NT-3 also reduced the blood glucose level in db/db mice. These results provide a novel insight that neurotrophin functions on the endocrine system as well as the nervous system.

Ciliary neurotrophic factor (CNTF) shares structural and functional properties with members of the hematopoietic cytokine family. It is composed of a four-helix bundle structure and shares the transmembrane signal transducing proteins, glycoprotein-130 (gp130) and leukemia inhibitory factor receptor (LIF-R). Structure-function analysis showed that the gp130-interactive proteins bind in a similar manner to that of growth hormone (sites I and II). In addition, gp130-interactive proteins and granulocyte colony-stimulating factor (G-CSF) utilize another binding site (site III) at the boundary between CD loop and helix D. CNTF triggers the association of receptor components, resulting in activation of a signal transduction cascade mediated by specific intracellular protein tyrosine kinases. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and Ras/mitogen-activated protein kinase (MAPK) signaling pathways have been characterized in terms of gp130-interactive protein, and there should be other pathways and some crosstalk between them to enhance, prolong, or specify the signals.

We have detected, isolated and purified an endonuclease from 5-fluoro-2′-deoxyuridine-treated FM3A cells. The molecular mass of the endonuclease was approximately 40 kDa as judged by sodium dodecyl sulfate-polyacrylamide DNA-containing gel electrophoresis. The endonuclease causes double strand breaks in DNA, with an optimum pH at 6.

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the alpha-helical cytokine family. hCNTF was subjected to three-dimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5- to 10-fold higher biological activity. The D1 cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the D1 cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to alpha-helical cytokine proteins reactive with gp130, a component protein of the receptors.

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.

We investigated the neuroprotective effects of brain-derived neurotrophic factor (BDNF) and its influence on the functional recovery of the retina following light-induced retinal damage by electroretinogram (ERG). Rats were exposed to constant fluorescent light for 2, 5, 7, or 14 days, then returned to a cyclic light environment for 14 days. The result indicated that BDNF had few effects on the a-wave amplitude, but there was a statistically significant difference in the b-wave amplitudes between BDNF-treated and control eyes from day 0-14 of the recovery period following 2 days of light exposure (p0.05). Our findings suggest that BDNF not only protects the retinal neuronal function but also enhances the recovery from retinal light damage.

Brain-derived neurotrophic factor (BDNF) shows neurotrophic effects on adult motor neurons when given systemically, But it is unknown whether systemically administered BDNF is transported to central cell bodies to affect them directly. Here we used immunohistochemistry to investigate the transport of peripherally injected BDNF to spinal motor neurons and the subsequent activation of a signaling pathway. We first injected BDNF into the flexor digitorum brevis (FDB) and analyzed the motor nucleus that projects to the FDB for BDNF immunoreactivity (BDNF-ir) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 immunoreactivity (pERK1/2-ir). Both immunoreactivities were observed in the motor neuron cell bodies. Next, BDNF was injected subcutaneously (s.c.) into rats with a unilaterally axotomized sciatic nerve. pERK1/2-ir was detected in motor neurons of the lesioned side. BDNF-ir and pERK1/2-ir were also observed on the unlesioned side when a high dose of BDNF was injected. Therefore, we examined BDNF-ir and pERK1/2-ir after injecting BDNF s.c. into normal rats. Both immunoreactivities were observed in motor nuclei on both sides. Finally, we examined pERK1/2-ir after a lower dose of BDNF was injected, which prevents the decrease in choline acetyl transferase that occurs in the motor neuron upon axotomy. Spinal motor nuclei contained a few cell bodies with pERK1/2-ir. These findings represent the first direct evidence that subcutaneously injected BDNF is transported to motor neurons and that it activates a signaling pathway in the spinal cord and exhibits neurotrophic effects in vivo.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been shown to be a potent and effective trophic factor for motor neurons and other neurons of the peripheral and central nervous. Little is known, however, about the relationship between the efficacy and pharmacokinetics of s.c. administered BDNF. In this study, the efficacy of BDNF on motor neuron protection in sciatic or facial nerve axotomy models was examined and compared with the concommitant concentrations of BDNF in plasma. Delayed treatment (started at 1 week after surgery) of BDNF was also shown to retard choline acetyltransferase reduction in sciatic nerve axotomy models.

Purpose: To determine if brain-derived neurotrophic factor (BDNF) has a neuroprotective effect against N -methyl- d -aspartate (NMDA)-induced cell death in retina. Methods: NMDA was injected into the vitreous of rat eyes. NMDA-induced neuronal death was measured by morphometric analyses on cell counts of ganglion cell layer cells and thickness of retinal layers. Also, we conducted additional experiment using retrograde labeling with a fluorescent tracer (Fluoro-Gold) for exact counting of retinal ganglion cells (RGCs). In addition, intravitreal glutamate levels were measured with the use of a high-performance liquid chromatography (HPLC) system. Results: Morphometric analysis of retinal damage in NMDA-injected eyes showed that BDNF could protect inner retinal cells from glutamate receptor-mediated neuronal death. Also, counts of RGCs labeled with a fluorescent tracer showed that BDNF could protect RGCs from glutamate receptor-mediated neuronal death. Furthermore, measurements of intravitreal glutamate levels indicated an increase in this excitatory amino acid in the vitreous after NMDA injection. Conclusions: Exogenous BDNF can protect inner retinal cells (possible RGCs and amacrine cells) from NMDA-induced neuronal death. However, increased intravitreal glutamate levels in response to NMDA-mediated neurotoxicity may augment retinal degeneration.

We previously reported that brain-derived neurotrophic factor (BDNF) regulates both food intake and blood glucose metabolism in rodent obese diabetic models such as C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice. To elucidate the effect of BDNF on glucose metabolism, we designed a novel pellet pair-feeding apparatus to eliminate the effect of appetite alteration on glucose metabolism. The apparatus was used to synchronize food intake precisely between BDNF-treated and vehicle-treated db/db mice. It was shown using this pellet pair-feeding apparatus that BDNF administered daily (20 mg x kg(-1) x day(-1)) to db/db mice significantly lowered blood glucose compared with pellet pair-fed db/db mice. To evaluate the effect of BDNF on insulin action, we used streptozotocin-induced type 1 diabetic mice. In this case, BDNF did not lower blood glucose concentration but rather enhanced the hypoglycemic action of insulin. In hyperglycemic db/db mice, pancreatic insulin content was reduced and glucagon content was increased compared with normoglycemic db/m mice. BDNF administered to db/db mice significantly restored both pancreatic insulin and glucagon content. Histological observations of aldehyde-fuchsin staining and immunostaining with anti-insulin indicated that insulin-positive pancreatic beta-cells were extensively regranulated by BDNF administration. We also studied the effect of BDNF on KK mice, normoglycemic animals with impaired glucose tolerance. In these mice, BDNF administration improved insulin resistance in the oral glucose tolerance test. To elucidate how blood glucose was metabolized in BDNF-treated animals, we investigated the effect of BDNF on the energy metabolism of db/db mice. Body temperature and oxygen consumption of the pellet pair-fed vehicle-treated mice were remarkably lower than the ad libitum-fed vehicle-treated mice. Daily BDNF administration for 3 weeks completely ameliorated both of the reductions. Finally, to clarify its action mechanism, the effect of intracerebroventricular administration of BDNF on db/db mice was examined. Here, a small dose of BDNF was found to be effective in lowering blood glucose concentration. This indicates that BDNF regulates glucose metabolism by acting directly on the brain.

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.

To assess the therapeutic potential of brain-derived neurotrophic factor (BDNF) in clinics, we extensively investigated the effects of BDNF on adult motor neurons in a rat spinal root avulsion model. Intrathecal administration of BDNF immediately after the spinal root avulsion greatly protected against the motor neuron cell death. BDNF also showed a protective effect on the atrophy of soma and on the reduction of transmitter-related enzymes such as choline acetyl transferase and acetylcholine esterase. Very interestingly, BDNF induced axonal outgrowth of severely damaged motor neurons at the avulsion site. The BDNF administration following 2-week treatment with phosphate-buffered saline after avulsion prevented further augmentation of cell death and reversed cholinergic transmitter-related enzyme deficiency. BDNF was demonstrated to possess a wide variety of biological effects on survival, soma size, cholinergic enzymes, and axonal outgrowth of adult motor neurons. These results provide a rationale for BDNF treatment in motor neuron diseases such as spinal cord injury and amyotrophic lateral sclerosis.

We have achieved high-level expression of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lymphoblastoid Namalwa cells by introducing and subsequently amplifying an expression vector encoding human GM-CSF and mouse dihydrofolate reductase (DHFR). Transformants expressing elevated levels of both GM-CSF and DHFR were selected by a step-wise increase in the concentration of methotrexate (MTX). Several cell lines resistant in 800 nM MTX have been established that express GM-CSF at a rate of 10-20 micrograms/10(6) cells/day. The amplified genes are integrated and stably maintained in the chromosomes of these cell lines. Analysis of the GM-CSF produced in the amplified Namalwa cells revealed that the molecular-size distribution due to varied degrees of glycosylation was similar to that observed in the naturally-occurring molecules.

Various 5-substituted 1-beta-D-arabinofuranosyluracil 5'-triphosphates (H, methyl, ethyl, n-propyl, n-butyl, (E)-bromovinyl, styryl, and beta-phenylethyl derivatives) were prepared and their inhibitory effects on two different herpes virus-induced DNA polymerases (OMV and HCMV) were studied. These dTTP analogues inhibited the incorporation of [3H]dTMP into DNA in vitro. Among them, analogues having a vinyl group at the 5-position were strongly active against DNA polymerases induced on herpes virus infection. Kinetic analysis showed that the inhibition by the analogues was essentially competitive with respect to the substrate, dTTP. The K1 values (microM) for AraUTP (2.4), AraTTP (1.0), BVAUTP (0.8), and StUAUTP (0.8) were smaller than the Km value (microM) for dTTP (3.4), but those for AraEtUTP, AraPrUTP, and AraBuUTP (5-14) were larger than the Km for dTTP in the case of HCMV-induced DNA polymerase. In contrast to these results, OMV-induced DNA polymerase seemed to be more resistant to these inhibitors than HCMV-induced DNA polymerase. However, the mode of the structure of substituent groups at the 5-position of base moieties is almost the same for the two DNA polymerases, except for in the case of AraUTP itself.

The newly synthesized araUTP and its derivatives such as ara5-ethyl UTP (1-β-D-arabinofuranosyl-5-ethyluracil triphosphate) and ara5-propyl UTP (l-β-D-arabinofuranosyl-5-propyluracil triphosphate) were tested for inhibition of DNA polymerase γ and of reverse transcriptase. All these three compounds as well as araTTP inhibited the activity of DNA polymerase γ. On the contrary, the activity of reverse transcriptase was inhibited by araUTP, but not by ara5-ethyl UTP and ara5-propyl UTP. The results indicate that there is a distinct difference in function between the active site of DNA polymerase γ and that of reverse transcriptase.

Various 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA synthesis in vitro. RNA polymerase I utilized the 5-methyl-, chloro, bromo, and iodo derivatives of UTP more efficiently than unmodified UTP, but RNA polymerase II utilized UTP most efficiently. 5-Methyl-4-thiouridine 5'-triphosphate (4-thio TTP) was utilized more efficiently than UTP by RNA polymerase I. On the other hand, it was found that 4-thio TTP was a selective substrate for RNA polymerase I and that its incorporation by RNA polymerase II was very slow. Thus recognition of UTP analogues as substrates by RNA polymerase I and II was different. These observation were attributed from kinetic analyses to differences in catalytic activity (Vmax).

Treatment of D-xylose (1) with 0.5% methanolic hydrogen chloride under controlled conditions followed by benzoylation and acetolysis afforded crystalline 1-O-acetyl-2, 3, 5-tri-O-benzoyl-α-D-xylofuranose (4) in good yield. Coupling of 4 with 2, 4-bis-trimethylsilyl derivatives of 5-alkyluracils (methyl, ethyl, propyl and butyl) (5a-5d), 5-fluorouracil (5e) and uracil (5f) in acetonitrile in the presence of stannic chloride gave 1-(2,3,5-tri-O-benzoyl-β-D-xylofuranosyl)-nucleosides (6a-6f). Saponification of 6 with sodium methoxide afforded 1-β-D-xylofuranosyl-5-substituted uracils (7a-7f). Condensation of 4 with free adenine in similar fashion and deblocking gave carcinostatic 9-β-D-xylofuranosyladenine (7g).

In order to determine the mode of action of cytostatic 9-beta-D-xylofuranosyladenine (xylo-A), the inhibitory effects of 9-beta-D-xylofuranosyladenine 5'-triphosphate (xylo-ATP) on DNA-dependent RNA polymerases I and II purified from cherry salmon (Oncorhynchus masou) liver nuclei were studied. This nucleotide showed strong inhibitory action on both RNA polymerases I and II. The K1 values are 14 microM for polymerase I and 5 microM for polymerase II (Km values of ATP are 37 microM for polymerase I and 40 microM for polymerase II). The mode of xylo-ATP was competitive with respect to the incorporation of AMP into RNA and non-competitive to UTP and CTP.

The pyrimidine 3'-deoxyriboside 5'-triphosphates, pyrimidine counterparts of cordycepin 5'-triphosphate, (1) were conveniently synthesized from cordycepin as potential deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase inhibitors. For the synthesis of 3'-deoxycytidine, p-chlororobenzoylation of 1 followed by acetolysis afforded an anomeric mixture of 1-O-acetyl-2, 5-di-O-p-chlorobenzoyl-3-deoxy-D-ribofuranose (3) in good yield. Condensation of 2, 4-bis-trimethylsilyl-N4-acylcytosine (4a, b) with 3 in the presence of stannic chloride gave fully acylated 3'-deoxycytidine (5) in excellent yield. The protecting group was removed to give 3'-deoxycytidine (6), which on phosphorylation gave 3'-deoxycytidine 5'-monophosphate (7), a key intermediate in the present study. Deamination of 7 with nitrous acid afforded 3'-deoxycytidine-5'-monophosphate (8). Additionally, 2, 4-bis-trimethylsilyloxy-5-methyl-or ethylpyrimidine (4c, d) was coupled with 3 in a similar manner to give fully acylated 5-methyl-(5c) or 5-ethyl-3'-deoxyuridine (5d). The 5'-monophosphates were converted to the corresponding 5'-triphosphates by the phosphoroimidazolidate method.

Various 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates (hydrogen, methyl-, ethyl-, n-propyl, n-butyl, fluoro-, chloro-, bromo-, and iodo derivatives) and some of the 3'-deoxyribofuranosyl nucleotides (3'-deoxy UTP and 3'-deoxy TTP) were synthesized chemically and their inhibitory effects on DNA-dependent RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied systematically. These 3'-modified UTP analogues could not be utilized as substrates in place of UTP, but they did inhibit the incorporation of UMP into RNA in vitro. In contrast, 2'-modified UTP analogues, such as 2'-dTTP and Ara TTP, were neither substrates nor inhibitors. Kinetic analysis showed that the inhibition by these compounds was essentially competitive with substrate UTP. The K1 values of RNA polymerase I for the analogues were smaller (2-6 microM) than the Km value for UTP (8 microM), but those for xylo-EtUTP, xylo-PrUTP, and xylo-BuUTP were larger (about 20 microM) than the Km for UTP. In contrast to these alkyl groups with steric and electron-donating effects, halogen groups have electron-withdrawing effects on the uracil nucleus. Therefore, it was concluded that the inhibitory activity of these analogues on RNA polymerase I was not affected by the inductive effects of substituent groups at the 5-position of uracil nucleus but by their steric effects. On the other hand, all of the K1 values of RNA polymerase II for UTP analogues were smaller (0.4-3 microM) than the Km value for UTP (4 microM). In this case, neither steric effect nor an inductive effect of substituents on UTP analogues influenced the inhibitory activity towards RNA polymerase II.

A continuous spectrophotometric assay for thymidine phosphorylase has been developed and used to analyze the substrate properties of a number of 5-substituted 2'-deoxyuridines. A QSAR was found which relates the catalytic efficiency for various substrates to a single variable, the inductive field constant F.