Synucleinopathies form a group of neurodegenerative diseases defined by the misfolding and aggregation of alpha-synuclein (alpha-syn). Abnormal accumulation and spreading of alpha-syn aggregates lead to synapse dysfunction and neuronal cell death. Yet, little is known about the synaptic mechanisms underlying the alpha-syn pathology. Here we identified beta-isoforms of neurexins (beta-NRXs) as presynaptic organizing proteins that interact with alpha-syn preformed fibrils (alpha-syn PFFs), toxic alpha-syn aggregates, but not alpha-syn monomers. Our cell surface protein binding assays and surface plasmon resonance assays reveal that alpha-syn PFFs bind directly to beta-NRXs through their N-terminal histidine-rich domain (HRD) at the nanomolar range (K(D): ~500 nM monomer equivalent). Furthermore, our artificial synapse formation assays show that alpha-syn PFFs diminish excitatory and inhibitory presynaptic organization induced by a specific isoform of neuroligin 1 that binds only beta-NRXs, but not alpha-isoforms of neurexins. Thus, our data suggest that alpha-syn PFFs interact with beta-NRXs to inhibit beta-NRX-mediated presynaptic organization, providing novel molecular insight into how alpha-syn PFFs induce synaptic pathology in synucleinopathies such as Parkinson's disease and dementia with Lewy bodies.
MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs) which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs. In crystal structures, the six immunoglobulin (Ig) and single fibronectin III (FN3) domains of MDGA1 reveal a striking compact, triangular shape, both alone and in complex with NLGNs. Whether this unusual domain arrangement is required for biological function or other arrangements occur with different functional outcomes is unknown. Here, we show that wild-type MDGA1 can adopt both compact and extended 3D conformations that bind NLGN2. Designer mutants targeting strategic molecular elbows in MDGA1 alter the distribution of 3D conformations while leaving the binding affinity between soluble ectodomains of MDGA1 and NLGN2 intact. In contrast, in a cellular context, these mutants result in unique combinations of functional consequences, including altered binding to NLGN2, decreased capacity to conceal NLGN2 from NRXN1beta, and/or suppressed NLGN2-mediated inhibitory presynaptic differentiation, despite the mutations being located far from the MDGA1:NLGN2 interaction site. Thus, the 3D conformation of the entire MDGA1 ectodomain appears critical for its function, and its NLGN-binding site on Ig1-Ig2 is not independent of the rest of the molecule. As a result, global 3D conformational changes to the MDGA1 ectodomain via strategic elbows may form a molecular mechanism to regulate MDGA1 action within the synaptic cleft.
The molecular pathological mechanisms underlying schizophrenia remain unclear; however, genomic analysis has identified genes encoding important risk molecules. One such molecule is neurexin 1alpha (NRXN1alpha), a presynaptic cell adhesion molecule. In addition, novel autoantibodies that target the nervous system have been found in patients with encephalitis and neurological disorders. Some of these autoantibodies inhibit synaptic antigen molecules. Studies have examined the association between schizophrenia and autoimmunity; however, the pathological data remain unclear. Here, we identified a novel autoantibody against NRXN1alpha in patients with schizophrenia (n=2.1%) in a Japanese cohort (n=387). None of the healthy control participants (n=362) were positive for anti-NRXN1alpha autoantibodies. Anti-NRXN1alpha autoantibodies isolated from patients with schizophrenia inhibited the molecular interaction between NRXN1alpha and Neuroligin 1 (NLGN1) and between NRXN1alpha and Neuroligin 2 (NLGN2). Additionally, these autoantibodies reduced the frequency of the miniature excitatory postsynaptic current in the frontal cortex of mice. Administration of anti-NRXN1alpha autoantibodies from patients with schizophrenia into the cerebrospinal fluid of mice reduced the number of spines/synapses in the frontal cortex and induced schizophrenia-related behaviors such as reduced cognition, impaired pre-pulse inhibition, and reduced social novelty preference. These changes were improved through the removal of anti-NRXN1alpha autoantibodies from the IgG fraction of patients with schizophrenia. These findings demonstrate that anti-NRXN1alpha autoantibodies transferred from patients with schizophrenia cause schizophrenia-related pathology in mice. Removal of anti-NRXN1alpha autoantibodies may be a therapeutic target for a subgroup of patients who are positive for these autoantibodies.
Chemical neurotransmission typically occurs through synapses. Previous ultrastructural examinations of monoamine neuron axon terminals often failed to identify a pre- and postsynaptic coupling, leading to the concept of "volume" transmission. Whether this results from intrinsic properties of these neurons remains undefined. We find that dopaminergic neurons in vitro establish a distinctive axonal arbor compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. While most dopaminergic varicosities are active and contain exocytosis proteins like synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in dopaminergic terminals that are in proximity to a target cell. Finally, we found that the proteins neurexin-1alpha(SS4-) and neuroligin-1(A+B) play a critical role in the formation of synapses by dopamine (DA) neurons. Our findings suggest that DA neurons are endowed with a distinctive developmental connectivity program.
Title: Synaptic Organizers in Alzheimer's Disease: A Classification Based on Amyloid-beta Sensitivity Lee AK, Khaled H, Chofflet N, Takahashi H Ref: Front Cell Neurosci, 14:281, 2020 : PubMed
Synaptic pathology is one of the major hallmarks observed from the early stage of Alzheimer's disease (AD), leading to cognitive and memory impairment characteristic of AD patients. Synaptic connectivity and specificity are regulated by multiple trans-bindings between pre- and post-synaptic organizers, the complex of which exerts synaptogenic activity. Neurexins (NRXs) and Leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are the major presynaptic organizers promoting synaptogenesis through their distinct binding to a wide array of postsynaptic organizers. Recent studies have shown that amyloid-beta oligomers (AbetaOs), a major detrimental molecule in AD, interact with NRXs and neuroligin-1, an NRX-binding postsynaptic organizer, to cause synaptic impairment. On the other hand, LAR-RPTPs and their postsynaptic binding partners have no interaction with AbetaOs, and their synaptogenic activity is maintained even in the presence of AbetaOs. Here, we review the current evidence regarding the involvement of synaptic organizers in AD, with a focus on Abeta synaptic pathology, to propose a new classification where NRX-based and LAR-RPTP-based synaptic organizing complexes are classified into Abeta-sensitive and Abeta-insensitive synaptic organizers, respectively. We further discuss how their different Abeta sensitivity is involved in Abeta vulnerability and tolerance of synapses for exploring potential therapeutic approaches for AD.
BACKGROUND: In recent years, systemic chemotherapy has significantly improved the prognosis of metastatic colorectal cancer (CRC); however, different patients have different responses to chemotherapeutics. METHODS: Dipeptidyl peptidase 9 (DPP9) is an enzyme in the dipeptidyl peptidase IV family that has been reported to increase drug sensitivity in acute myeloid leukemia. In this study, we examined the relationship between DPP9 expression and the prognosis of patients with CRC, as well as the role of DPP9 in anticancer drug resistance. Moreover, the effects of the DPP9 inhibitors talabostat and vildagliptin in CRC cell lines and primary cultured cells were assessed. RESULTS: High expression of DPP9 was associated with worse prognosis in 196 patients with CRC. Cell viability was markedly inhibited in CRC cell lines transfected with DPP9 small interfering RNA or small hairpin RNA. Talabostat suppressed proliferation in CRC cell lines and primary cultured cells, and increased their sensitivity to chemotherapy. Vildagliptin, a DPP family inhibitor currently administered for diabetes, also increased the sensitivity of CRC cells to anticancer drugs. CONCLUSION: DPP9 was a poor prognostic factor for CRC and could be a new therapeutic target, while vildagliptin could be used as a repositioned drug for CRC treatment.
PURPOSE: Acyl ghrelin is an orexigenic peptide. Active ghrelin ratio, the ratio of acyl ghrelin to total ghrelin, has an important role in physiological functions and gastrointestinal symptoms. However, low active ghrelin ratio-related characteristics, gastrointestinal symptoms, and chemotherapy-induced gastrointestinal toxicity in patients with advanced pancreatic cancer have not been previously evaluated. The goal of this study was to identify low active ghrelin ratio-related factors in treatment-naive advanced pancreatic cancer patients. METHODS: Patients with treatment-naive advanced pancreatic cancer were eligible for inclusion in this study. Active ghrelin ratio and clinical parameters of patients were prospectively recorded. Factors correlated with low active ghrelin ratio and survival were analyzed. RESULTS: In total, 92 patients were analyzed. Low active ghrelin ratio-related factors were advanced age (P < 0.01), severe appetite loss (P < 0.01), and decreased cholinesterase (P < 0.01). The adverse events of grade 2 or higher anorexia tended to increase in patients with low active ghrelin ratio. However, no differences were found in survival and body composition between low and high active ghrelin ratio groups. CONCLUSIONS: Low active ghrelin ratio was related to lack of appetite and low cholinesterase and tended to be related to anorexia grade 2 or higher in patients with treatment-naive advanced pancreatic cancer.
Title: Emerging roles of the neurotrophin receptor TrkC in synapse organization Naito Y, Lee AK, Takahashi H Ref: Neurosci Res, 116:10, 2017 : PubMed
Tropomyosin-receptor-kinase (Trk) receptors have been extensively studied for their roles in kinase-dependent signaling cascades in nervous system development. Synapse organization is coordinated by trans-synaptic interactions of various cell adhesion proteins, a representative example of which is the neurexin-neuroligin complex. Recently, a novel role for TrkC as a synapse organizing protein has been established. Post-synaptic TrkC binds to pre-synaptic type-IIa receptor-type protein tyrosine phosphatase sigma (PTPsigma). TrkC-PTPsigma specifically induces excitatory synapses in a kinase domain-independent manner. TrkC has distinct extracellular domains for PTPsigma- and NT-3-binding and thus may bind both ligands simultaneously. Indeed, NT-3 enhances the TrkC-PTPsigma interaction, thus facilitating synapse induction at the pre-synaptic side and increasing pre-synaptic vesicle recycling in a kinase-independent fashion. A crystal structure study has revealed the detailed structure of the TrkC-PTPsigma complex as well as competitive modulation of TrkC-mediated synaptogenesis by heparan sulfate proteoglycans (HSPGs), which bind the same domain of TrkC as PTPsigma. Thus, there is strong evidence supporting a role for the TrkC-PTPsigma complex in mechanisms underlying the fine turning of neural connectivity. Furthermore, disruption of the TrkC-PTPsigma complex may be the underlying cause of certain psychiatric disorders caused by mutations in the gene encoding TrkC (NTRK3), supporting its role in cognitive functions.
BACKGROUND: Trelagliptin, an oral DPP-4 inhibitor, which is administered once per week and characterized by a long half-life in blood. The effects of trelagliptin on vascular endothelial functions have not been clarified to date. The objective of the present study was to examine the effects of trelagliptin on vascular endothelial functions in patients with type 2 diabetes mellitus (DM) using flow-mediated dilatation (FMD), adiponectin, and asymmetric dimethylarginine (ADMA) as evaluation indicators. METHODS: This study was a preliminary single-arm prospective pilot study. The subjects of this study were type 2 DM patients aged 20-74 years, who visited our outpatient department. The patients were treated with trelagliptin, and their FMD, adiponectin, and ADMA levels were measured at baseline and at 12 weeks after initial treatment to determine the changes during the study period. RESULTS: A total of 27 patients, excluding three dropouts, were included in the population for analysis. Trelagliptin treatment showed no significant changes in FMD (2.42 +/- 2.7% at baseline vs. 2.66 +/- 3.8% post-treatment, P = 0.785) and ADMA (0.41 +/- 0.0 microg/mL at baseline vs. 0.40 +/- 0.0 microg/mL post-treatment, P = 0.402). Trelagliptin treatment resulted in a significant increase of serum adiponectin level (7.72 +/- 6.9 microg/mL at baseline vs. 8.82 +/- 8.3 microg/mL post-treatment, P < 0.002). CONCLUSIONS: In this pilot study, trelagliptin treatment showed no significant changes in FMD. On the other hand, it was believed that trelagliptin treatment may increase serum adiponectin level. Trial Registration http://www.umin.ac.jp (Trial ID UMIN000018311).
Title: Efficacy and safety of teneligliptin in addition to insulin therapy in type 2 diabetes mellitus patients on hemodialysis evaluated by continuous glucose monitoring Yajima T, Yajima K, Hayashi M, Takahashi H, Yasuda K Ref: Diabetes Res Clin Pract, 122:78, 2016 : PubMed
AIMS: Appropriate glycemic control without hypoglycemia is important in patients with type 2 diabetes on hemodialysis. Teneligliptin, a novel dipeptidyl peptidase-4 inhibitor, can be used without dose adjustment for these patients. Using continuous glucose monitoring (CGM), we evaluated the efficacy and safety of adding teneligliptin to insulin therapy. METHODS: Twenty-one type 2 diabetes mellitus patients on hemodialysis treated with insulin were enrolled. After the adjustment of insulin dose, their blood glucose level was monitored by CGM. Insulin dose was reduced after teneligliptin administration. RESULTS: The median total daily insulin dose significantly reduced from 18 (9-24)U to 6 (0-14)U (p<0.0001). Maximum, mean, and standard deviation of blood glucose level on the hemodialysis and non-hemodialysis days did not change after teneligliptin administration. However, minimum blood glucose level was significantly elevated on the hemodialysis day after teneligliptin administration (from 3.9+/-1.0mmol/L to 4.4+/-0.9mmol/L, p=0.040). The incidence of asymptomatic hypoglycemia on the hemodialysis day detected by CGM significantly decreased from 38.1% to 19.0% (p=0.049). CONCLUSIONS: Teneligliptin may contribute toward reducing the total daily insulin dose and preventing hypoglycemic events on the hemodialysis day in type 2 diabetes mellitus patients.
The incidence of aspergillosis by Aspergillus infection has dramatically increased in recent years. Aspergillus udagawae, a species related to Aspergillus fumigatus, is known as an emerging pathogen of aspergillosis. Here, we present the draft genome sequence of A. udagawae strain IFM 46973(T).
Title: Genetic distance in the whole-genome perspective on Listeria monocytogenes strains F2-382 and NIHS-28 that show similar subtyping results Kyoui D, Takahashi H, Miya S, Kuda T, Igimi S, Kimura B Ref: BMC Microbiol, 14:309, 2014 : PubMed
BACKGROUND: Genome subtyping approaches could provide useful epidemiological information regarding food pathogens. However, the full genomic diversity of strains that show similar subtyping results has not yet been completely explored. Most subtyping methods are based on the differences of only a portion of the genome. We investigated two draft genome sequences of Listeria monocytogenes strain F2-382 and NIHS-28, which have been identified as closely related strains by subtyping (identical multi-virulence-locus sequence typing and multiple-locus variable number tandem repeat analysis sequence types and very similar pulsed-field gel electrophoresis patterns), despite their different sources. RESULTS: Two closely related strains were compared by genome structure analysis, recombination analysis, and single nucleotide polymorphism (SNP) analysis. Both genome structure analysis and recombination analysis showed that these two strains are more closely related than other strains, from a whole-genome perspective. However, the analysis of SNPs indicated that the two strains differ at the single nucleotide level. CONCLUSION: We show the relationship between the results of genome subtyping and whole-genome sequencing. It appears that the relationships among strains indicated by genome subtyping methods are in accord with the relationships indicated by whole-genome analysis. However, our results also indicate that the genetic distance between the closely related strains is greater than that between clonal strains. Our results demonstrate that subtyping methods using a part of the genome are reliable in assessing the genetic distance of the strains. Furthermore, the genetic differences in the same subtype strains may provide useful information to distinguish the bacterial strains.
AIM: To elucidate the correlation between hepatic blood flow and liver function in alcoholic liver cirrhosis (AL-LC). METHODS: The subjects included 35 patients with AL-LC (34 men, 1 woman; mean age, 58.9 +/- 10.7 years; median age, 61 years; range: 37-76 years). All patients were enrolled in this study after obtaining written informed consent. Liver function was measured with tests measuring albumin (Alb), prothrombin time (PT), brain natriuretic peptide (BNP), branched amino acid and tyrosine ratio (BTR), branched chain amino acid (BCAA), tyrosine, ammonia (NH3), cholinesterase (ChE), immunoreactive insulin (IRI), total bile acid (TBA), and the retention rate of indocyanine green 15 min after administration (ICG R15). Hepatic blood flow, hepatic arterial tissue blood flow (HATBF), portal venous tissue blood flow (PVTBF), and total hepatic tissue blood flow (THTBF) were simultaneously calculated using xenon computed tomography. RESULTS: PVTBF, HATBF and THTBF were 30.2 +/- 10.4, 20.0 +/- 10.7, and 50.3 +/- 14.9 mL/100 mL/min, respectively. Alb, PT, BNP, BTR, BCAA, tyrosine, NH3, ChE, IRI, TBA, and ICG R15 were 3.50 +/- 0.50 g/dL, 72.0% +/- 11.5%, 63.2 +/- 56.7 pg/mL, 4.06 +/- 1.24, 437.5 +/- 89.4 mumol/L, 117.7 +/- 32.8 mumol/L, 59.4 +/- 22.7 mug/dL, 161.0 +/- 70.8 IU/L, 12.8 +/- 5.0 mug/dL, 68.0 +/- 51.8 mumol/L, and 28.6% +/- 13.5%, respectively. PVTBF showed a significant negative correlation with ICG R15 (r = -0.468, P <0.01). No significant correlation was seen between ICG 15R, HATBF and THTBF. There was a significant correlation between PVTBF and Alb (r = 0.2499, P < 0.05), and NH3 tended to have an inverse correlation with PVTBF (r = -0.2428, P = 0.0894). There were also many significant correlations between ICG R15 and liver function parameters, including Alb, NH3, PT, BNP, TBA, BCAA, and tyrosine (r = -0.2156, P < 0.05; r = 0.4318, P < 0.01; r = 0.4140, P < 0.01; r = 0.3610, P < 0.05; r = 0.5085, P < 0.001; r = 0.4496, P < 0.01; and r = 0.4740, P < 0.05, respectively). CONCLUSION: Our investigation showed that there is a close correlation between liver function and hepatic blood flow.
Title: Interaction between autism-linked MDGAs and neuroligins suppresses inhibitory synapse development Pettem KL, Yokomaku D, Takahashi H, Ge Y, Craig AM Ref: Journal of Cell Biology, 200:321, 2013 : PubMed
Rare variants in MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors), including multiple protein-truncating deletions, are linked to autism and schizophrenia, but the function of these genes is poorly understood. Here, we show that MDGA1 and MDGA2 bound to neuroligin-2 inhibitory synapse-organizing protein, also implicated in neurodevelopmental disorders. MDGA1 inhibited the synapse-promoting activity of neuroligin-2, without altering neuroligin-2 surface trafficking, by inhibiting interaction of neuroligin-2 with neurexin. MDGA binding and suppression of synaptogenic activity was selective for neuroligin-2 and not neuroligin-1 excitatory synapse organizer. Overexpression of MDGA1 in cultured rat hippocampal neurons reduced inhibitory synapse density without altering excitatory synapse density. Furthermore, RNAi-mediated knockdown of MDGA1 selectively increased inhibitory but not excitatory synapse density. These results identify MDGA1 as one of few identified negative regulators of synapse development with a unique selectivity for inhibitory synapses. These results also place MDGAs in the neurexin-neuroligin synaptic pathway implicated in neurodevelopmental disorders and support the idea that an imbalance between inhibitory and excitatory synapses may contribute to these disorders.
We implemented a two-step approach to detect potential predictor gene variants for neuroleptic-induced tardive dyskinesia (TD) in schizophrenic subjects. First, we screened associations by using a genome-wide (Illumina HumanHapCNV370) SNP array in 61 Japanese schizophrenia patients with treatment-resistant TD and 61 Japanese schizophrenia patients without TD. Next, we performed a replication analysis in 36 treatment-resistant TD and 138 non-TD subjects. An association of an SNP in the DPP6 (dipeptidyl peptidase-like protein-6) gene, rs6977820, the most promising association identified by the screen, was significant in the replication sample (allelic P=0.008 in the replication sample, allelic P=4.6 x 10(-6), odds ratio 2.32 in the combined sample). The SNP is located in intron-1 of the DPP6 gene and the risk allele was associated with decreased DPP6 gene expression in the human postmortem prefrontal cortex. Chronic administration of haloperidol increased Dpp6 expression in mouse brains. DPP6 is an auxiliary subunit of Kv4 and regulates the properties of Kv4, which regulates the activity of dopaminergic neurons. The findings of this study indicate that an altered response of Kv4/DPP6 to long-term neuroleptic administration is involved in neuroleptic-induced TD.
Mycobacterium avium complex (MAC) infection causes disseminated disease in immunocompromised hosts, such as human immunodeficiency virus (HIV)-positive patients, and pulmonary disease in persons without systemic immunosuppression, which has been increasing in many countries. In Japan, the incidence of pulmonary MAC disease caused by M. avium is about 7 times higher than that caused by M. intracellulare. To explore the bacterial factors that affect the pathological state of MAC disease caused by M. avium, we determined the complete genome sequence of the previously unreported M. avium subsp. hominissuis strain TH135 isolated from a HIV-negative patient with pulmonary MAC disease and compared it with the known genomic sequence of M. avium strain 104 derived from an acquired immunodeficiency syndrome patient with MAC disease. The genome of strain TH135 consists of a 4,951,217-bp circular chromosome with 4,636 coding sequences. Comparative analysis revealed that 4,012 genes are shared between the two strains, and strains TH135 and 104 have 624 and 1,108 unique genes, respectively. Many strain-specific regions including virulence-associated genes were found in genomes of both strains, and except for some regions, the G+C content in the specific regions was low compared with the mean G+C content of the corresponding chromosome. Screening of clinical isolates for genes located in the strain-specific regions revealed that the detection rates of strain TH135-specific genes were relatively high in specimens isolated from pulmonary MAC disease patients, while, those of strain 104-specific genes were relatively high in those from HIV-positive patients. Collectively, M. avium strains that cause pulmonary and disseminated disease possess genetically distinct features, and it suggests that the acquisition of specific genes during strain evolution has played an important role in the pathological manifestations of MAC disease.
Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABAA receptor- and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABAA receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2.
Distigmine bromide is a cholinesterase inhibitor widely used for the treatment of hypotonic neurogenic bladder. However, this drug is also known to cause cholinergic crisis, a rare but serious adverse reaction. Cholinergic crisis is an excessive amount of acetylcholine due to the systemic inhibition of cholinesterase activity, characterized by parasympathetic symptoms such as sweating, salivation, miosis, bradycardia, diarrhea and circulatory and respiratory failure. The incidence of cholinergic crisis has been estimated at approximately 0.2%, and the majority of the patients are elderly with underlying conditions such as cerebrovascular disease. Since 2004, we have encountered 5 cases of acute respiratory failure associated with cholinergic crisis induced by the administration of a normal oral dose of distigmine. We present these cases here and review an additional 23 cases from the literature in Japan. In these 28 cases, mechanical ventilation was required for 57%, with a mean duration of 5.1 days and a mortality rate of 11%. Pneumonia was observed in half of the cases in the acute phase, and relapse due to the readministration of distigmine was reported in 20% of cases. It is important to remember that cholinergic crisis in the elderly is often misdiagnosed and is occasionally treated as simple aspiration pneumonia.
Tardive dyskinesia (TD) is characterized by repetitive, involuntary, and purposeless movements that develop in patients treated with long-term dopaminergic antagonists, usually antipsychotics. By a genome-wide association screening of TD in 50 Japanese schizophrenia patients with treatment-resistant TD and 50 Japanese schizophrenia patients without TD (non-TD group) and subsequent confirmation in independent samples of 36 treatment-resistant TD and 136 non-TD subjects, we identified association of a single nucleotide polymorphism, rs2445142, (allelic p=2 x 10(-5)) in the HSPG2 (heparan sulfate proteoglycan 2, perlecan) gene with TD. The risk allele was significantly associated with higher expression of HSPG2 in postmortem human prefrontal brain (p<0.01). Administration of daily injection of haloperidol (HDL) for 50 weeks significantly reduced Hspg2 expression in mouse brains (p<0.001). Vacuous chewing movements (VCMs) induced by 7-week injection of haloperidol-reserpine were significantly infrequent in adult Hspg2 hetero-knockout mice compared with wild-type littermates (p<0.001). Treatment by the acetylcholinesterase inhibitor, physostigmine, was significantly effective for reduction of VCMs in wild-type mice but not in Hspg2 hetero-knockout mice. These findings suggest that the HSPG2 gene is involved in neuroleptic-induced TD and higher expression of HSPG2, probably even after antipsychotic treatment, and may be associated with TD susceptibility.
Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.
Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl), both with regard to localization and function: Cdc42-palm concentrates in dendritic spines and has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.
Ralstonia solanacearum 8107 (8107) is non-pathogenic to tobacco and elicits the hypersensitive response (HR). In Nicotiana tabacum cv. Samsun NN leaves infiltrated with 8107, acquired resistance to challenging tobacco mosaic virus (TMV) was induced 2-6 d after 8107-infiltration. hsr203J and hin1 genes were expressed only in the 8107-infiltrated area. On the other hand, the expression of PR-1a and PR-1b genes was not detected in the 8107-infiltrated area, but in areas other than that developing the HR. Expression of these PR-1 genes was regulated simultaneously and the kinetics of the expression was dependent on the distance from the infiltration area. Therefore, diffusible signal(s) might be produced in HR-causing cells and transmitted to peripheral cells resulting in expression of PR genes. In NahG10 tobacco infiltrated with 8107, the HR was induced but resistance to TMV was not. Analysis using NahG10 tobacco also showed that the salicylic acid (SA)-dependent signal regulated the expression of hsr203J and PR-1a, but not that of hin1 and PR-1b. These results suggest that resistance of tobacco to 8107 is SA-independent and involves a quite different mechanism from acquired resistance to TMV induced by 8107-infiltration which is SA-dependent.
Title: A carbamate-type cholinesterase inhibitor 2-sec-butylphenyl N-methylcarbamate insecticide blocks L-type ca2+ channel in guinea pig ventricular myocytes Futagawa H, Takahashi H, Nagao T, Adachi-Akahane S Ref: Japanese Journal of Pharmacology, 90:12, 2002 : PubMed
2-sec-Butylphenyl N-methylcarbamate (BPMC) is a carbamate-type cholinesterase (ChE) inhibitor with unique toxicological properties such as noncholinergic cardiovascular collapse. Effects of BPMC on L-type Ca2+ channel currents (ICa(L)) were studied in isolated guinea pig ventricular myocytes using the whole-cell patch-clamp technique, since the examination of cardiovascular responses indicated its Ca2+ antagonistic action. BPMC induced bradycardic and hypotensive responses in vivo and inhibited contraction of isolated papillary muscles (IC50 = 1.3 x 10(-4) M) in guinea pigs. BPMC produced reversible block of ICa(L) in the concentration range of 10(-4) - 10(-3) M. At test potentials between -30 mV and +20 mV, BPMC at 3 x 10(-4) M caused marked acceleration of decay rate of ICa(L) with moderate reduction of peak ICa(L) amplitude. BPMC (3 x 10(-4) M) shifted the steady-state inactivation curve to the hyperpolarizing direction by 12.7 mV. Decay rate of Ba2+ currents (IBa(L)) was also accelerated by BPMC. Fitting analysis of inactivation kinetics of IBa(L) with a two-exponential equation revealed that BPMC accelerates the slow inactivation component. At concentrations for blocking peak IBa(L) by ca. 30%, the inactivation kinetics of IBa(L) were significantly accelerated by BPMC, but merely slightly accelerated by Ca2+ channel antagonists such as diltiazem, nifedipine, or verapamil. These results indicate that BPMC, in addition to the inhibition of ChE, blocks L-type Ca2+ channels by accelerating voltage-dependent inactivation.
Title: Hydrotropism in abscisic acid, wavy, and gravitropic mutants of Arabidopsis thaliana Takahashi N, Goto N, Okada K, Takahashi H Ref: Planta, 216:203, 2002 : PubMed
We have developed experimental systems to study hydrotropism in seedling roots of Arabidopsis thaliana (L.) Heynh. Arabidopsis roots showed a strong curvature in response to a moisture gradient, established by applying 1% agar and a saturated solution of KCl or K(2)CO(3) in a closed chamber. In this system, the hydrotropic response overcame the gravitropic response. Hydrotropic curvature commenced within 30 min and reached 80-100 degrees within 24 h of hydrostimulation. When 1% agar and agar containing 1 MPa sorbitol were placed side-by-side in humid air, a water potential gradient formed at the border between the two media. Although the gradient changed with time, it still elicited a hydrotropic response in Arabidopsis roots. The roots curved away from 0.5-1.5 MPa of sorbitol agar. Various Arabidopsis mutants were tested for their hydrotropic response. Roots of aba1-1 and abi2-1 mutants were less sensitive to hydrotropic stimulation. Addition of abscisic acid restored the normal hydrotropic response in aba1-1 roots. In comparison, mutants that exhibit a reduced response to gravity and auxin, axr1-3 and axr2-1, showed a hydrotropic response greater than that of the wild type. Wavy mutants, wav2-1 and wav3-1, showed increased sensitivity to the induction of hydrotropism by the moisture gradient. These results suggest that auxin plays divergent roles in hydrotropism and gravitropism, and that abscisic acid plays a positive role in hydrotropism. Furthermore, hydrotropism and the wavy response may share part of a common molecular pathway controlling the directional growth of roots.
Title: The role of cholinergic and noncholinergic mechanisms in the cardiorespiratory failure produced by N-methylcarbamate cholinesterase inhibitors in rabbits Futagawa H, Kakinuma Y, Takahashi H Ref: Toxicol Appl Pharmacol, 165:27, 2000 : PubMed
We investigated the relative contribution of several cardiorespiratory components to acute lethality produced by N-methylcarbamate cholinesterase (ChE) inhibitors physostigmine, 2-sec-butylphenyl methylcarbamate (BPMC), and 2-isopropoxyphenyl methylcarbamate (PHC) in halothane-anesthetized rabbits. Intravenous injection of these compounds produced dose-dependent pressor and/or depressor responses related to each compound. A lethal dose of physostigmine resulted in cardiovascular collapse after a pressor response. That of PHC produced cardiovascular collapse after biphasic effects on blood pressure, a transient decrease followed by an increase. Unlike these compounds, BPMC elicited a rapidly developing depressor response followed by cardiovascular collapse. Artificial ventilation prevented cardiovascular collapse and lethal actions to physostigmine and PHC, but not BPMC. A degree of acute lethality to physostigmine and PHC depended on their anti-ChE activity, whereas BPMC exhibited a low degree of lethality relative to its anti-ChE activity. While the pressor response to physostigmine and PHC was ascribed to an atropine-sensitive increase in cardiac contractility, the depressor response to PHC and BPMC was attributed to an atropine-insensitive decrease in cardiac contractility and/or vascular resistance. Similar to the order for eliciting the depressor response in vivo, all three compounds inhibited contraction of the isolated cardiac and aortic smooth muscles with the order of their inhibition in terms of anti-ChE activity, i.e., BPMC > PHC > physostigmine. Thus, the primary cause of death with physostigmine and PHC is respiratory arrest subsequent to ChE inhibition, whereas BPMC exhibiting the low degree of lethality causes cardiovascular collapse mediated through direct inhibitory effects on cardiac and vascular smooth muscle contraction.
Title: CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli Takahashi H, Inada T, Postma P, Aiba H Ref: Molecular & General Genetics, 259:317, 1998 : PubMed
The cellular cAMP level is markedly down-regulated by cAMP receptor protein (CRP) in Escherichia coli. CRP regulates adenylate cyclase both at the level of transcription of its structural gene cya and at the level of enzyme activity. We established a method to determine the phosphorylation state of IIA(Glc), the glucose-specific phosphotransferase protein, in intact cells. We found that IIA(Glc) exists predominantly in the unphosphorylated form in wild-type cells growing in LB medium, while it is largely phosphorylated in crp or cya cells. Disruption of the ptsG gene that codes for the membrane component of the major glucose transporter (IICB(Glc)), and/or the fruF gene coding for FPr (fructose-specific hybrid phosphotransferase protein), did not affect the phosphorylation state of IIA(Glc). When IICB(Glc) was overproduced in the presence of glucose, the levels of both cAMP and phosphorylated IIA(Glc) in crp cells were concomitantly decreased to wild-type levels. In addition, when His-90 in IIA(Glc) was replaced by glutamine, both phosphorylation of IIA(Glc) and the overproduction of cAMP in crp cells were eliminated. We also found that extracts of crp+ cells markedly stimulate dephosphorylation of IIA(Glc)-P in vitro. We conclude that CRP-cAMP down-regulates adenylate cyclase primarily by reducing the level of phosphorylated IIA(Glc). The data suggest that unspecified proteins whose expression is under the control of CRP-cAMP are responsible for this regulation.
Title: Cardiovascular collapse through non-cholinergic mechanism after intravenous injection of N-methylcarbamate insecticide in rabbits Futagawa H, Kakinuma Y, Takahashi H Ref: Toxicology, 117:163, 1997 : PubMed
This study was undertaken to investigate whether cholinesterase (ChE) inhibitor exerts cardiovascular collapse through non-cholinergic mechanism in halothane-anesthetized rabbits. Physostigmine and N-methylcarbamate insecticides (BPMC = 2-sec-butylphenyl methylcarbamate and PHC = propoxur = 2-isopropoxyphenyl methylcarbamate) were employed as ChE inhibitors. Intravenous injection of physostigmine produced a dose-related pressor response a few minutes after the injection. In contrast, the injection of BPMC elicited a dose-related depressor response during the injection. PHC produced a slight depressor response during the injection followed by a dose-dependent pressor response. Norepinephrine (NE)-induced pressor response was inhibited by the ChE inhibitors with the same order and magnitude as the depressor response. ECG of physostigmine or PHC was characterized by an increase in QRS voltage and a sinus bradycardia, and that of BPMC by a decrease in QRS voltage. Atropine pretreatment inhibited the pressor response, the increase in QRS voltage and the sinus bradycardia, but not the depressor response and the decrease in QRS voltage. From these observations, it is suggested that the pressor response is ascribed to the cholinergic mechanism (acetylcholine accumulation through ChE inhibition), but the depressor response may result from a non-cholinergic mechanism. It is also suggested that the difference in the cardiovascular response is determined by a balance between cholinergic and non-cholinergic activity of each ChE inhibitor.
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
Title: Sequence and analysis of a 31 kb segment of the Bacillus subtilis chromosome in the area of the rrnH and rrnG operons. Liu H, Haga K, Yasumoto K, Ohashi Y, Yoshikawa H, Takahashi H Ref: Microbiology, 143:2763, 1997 : PubMed
Title: Effects of 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1 - H-1-benzazepin-8-yl)-1-propanone fumarate (TAK-147), a novel acetylcholinesterase inhibitor, on impaired learning and memory in animal models Miyamoto M, Takahashi H, Kato K, Hirai K, Ishihara Y, Goto G Ref: Journal of Pharmacology & Experimental Therapeutics, 277:1292, 1996 : PubMed
We examined the effects of p.o. administered 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-1-b enzazepin-8- yl)-1-propanone fumarate (TAK-147), a novel AChE inhibitor, on impaired learning and memory in animal models. At 1 to 3 mg/kg, TAK-147 ameliorated the passive avoidance deficit induced by diazepam. TAK-147 did not affect delayed-matching-to-position (DMTP) performance of normal rats at doses of 1 to 30 mg/kg assessed by using a three-lever operant chamber, but 9-amino-tetrahydroacridine disrupted the DMTP response at 5 to 20 mg/kg. Scopolamine (0.02-0.1 mg/kg s.c.) impaired DMTP performance, whereas methylscopolamine did not affect the DMTP task. TAK-147 ameliorated the impairment of DMTP performance induced by scopolamine without affecting the general behavior of the rats; however, 9-amino-tetrahydroacridine produced no significant amelioration of the impairment. The intraventricular injection of AF64A disrupted differential-reinforcement-of-low-rate 10-sec performance in rats, as demonstrated by marked decreases in reinforcement rate and response efficiency. TAK-147 slightly increased the reinforcement rate in AF64A-treated rats at a low dose of 1 mg/kg, but the effect was not significant statistically. TAK-147 had no significant effect on the duration of immobility in rats in a forced swimming test at doses of 2 to 10 mg/kg. 9-Amino-tetrahydroacridine prolonged the duration of immobility at 5 to 20 mg/kg. Furthermore, TAK-147 reversed reserpine-induced hypothermia and ptosis in mice at doses of 3 to 10 mg/kg, a result that implies an antidepressant-like action. These results indicate that TAK-147 ameliorates learning and memory impairment in animal models without affecting the general behavior or causing behavioral depression and suggest that TAK-147 may be useful for the treatment of Alzheimer's disease.
Effect of TAK-147, a novel acetylcholinesterase (AChE) inhibitor, on cerebral energy metabolism was investigated using an in vivo 31P-magnetic resonance spectroscopy (31P-MRS) technique and the autoradiographic 2-deoxy-[14C]-D-glucose method in aged Fischer 344 rats. We revealed that high-energy phosphate metabolites, phosphocreatine (PCr) and ATP, in the brain decreased gradually with aging and that significant decrement of cerebral PCr and ATP was observed from 13- and 8.5-month-old in comparison with those of 2.5-month-old rats, respectively. Daily oral administration of TAK-147 (1 mg/kg) for 40 days increased PCr and ATP levels in aged rats (29-month-old). To determine the site at which TAK-147 acts to increase high-energy phosphate metabolism, we investigated the rate of local cerebral glucose utilization (LCGU) in various brain regions. The rate of LCGU decreased in almost all brain regions in aged rats (28 months of age), and the decrease was significant in 29 out of the 35 regions. When TAK-147 was administered orally to the aged rats, the levels were dose dependently increased, especially in the auditory cortex. These results indicate that TAK-147 increases cerebral energy metabolism in aged rats.
The 49630 bp spo0H-rrnH region of the Bacillus subtilis genome has been fully sequenced. The sequence contains one partial and 62 complete ORFs, one partial and three complete rRNA genes and a cluster of six tRNA genes. The direction of the transcription and translation of 61 ORFs is the same as that of the movement of the replication fork. A homology search of 40 ORFs in newly determined sequence revealed that 27 of them had significant similarity to known proteins such as elongation factor G, elongation factor Tu, pseudouridine synthase I and ribsosomal proteins. Two adjacent genes, ybaD and ybaE, appeared to encode proteins belonging to the ATP-binding cassette (ABC) family.
Title: Non-cholinergic lethality following intravenous injection of carbamate insecticide in rabbits Takahashi H, Kakinuma Y, Futagawa H Ref: Toxicology, 93:195, 1994 : PubMed
This study was undertaken to investigate the possibility that non-cholinergic mechanism accounts for acute lethality of cholinesterase (ChE) inhibitor. Physostigmine and carbamate insecticides (2-s-butylphenyl methylcarbamate (BPMC); isoprocarb, 2-isopropylphenyl methylcarbamate (MIPC); and propoxur, 2-isopropoxyphenyl methylcarbamate (PHC)) were employed as ChE inhibitors. Rabbits intravenously given any of the ChE inhibitors showed typical signs of anti-ChE poisoning, a marked inhibition of systemic ChE activity, and an increase in RR interval on an ECG. Injection of physostigmine or PHC at a lethal dose produced a pressor response before cessation of spontaneous breathing. In contrast, injection of MIPC or BPMC primarily elicited a cardiovascular collapse, characterized by a rapid and progressive decrease in blood pressure and a decrease in QRS amplitude of ECG, before cessation of spontaneous breathing. The atropine pretreatment inhibited the pressor response, but not the depressor response and the QRS change. The pretreatment antagonized acute lethality of the ChE inhibitors except BPMC. It is suggested that the mode of lethality for intravenous ChE inhibitor could be determined by a balance between anti-ChE activity and some mechanism other than ChE inhibition. BPMC produced acute lethality through the latter mechanism rather than the former one.
Title: Pretreatment of rats with an organophosphorus insecticide, chlorfenvinphos, protects against subsequent challenge with the same compound Ikeda T, Kojima T, Yoshida M, Takahashi H, Tsuda S, Shirasu Y Ref: Fundamental & Applied Toxicology, 14:560, 1990 : PubMed
A single oral pretreatment of rats with chlorfenvinphos (CVP) reduced toxicity of the same compound subsequently administered. This protection occurred 8 hr and became maximal 24 hr after the oral pretreatment at a dose of 15 mg/kg (about half of its LD50). The 24-hr pretreatment with CVP increased the LD50 of CVP threefold, but did not change the type of toxic signs and time to death caused by CVP. The CVP pretreatment did not appreciably change the toxicities of the cholinergic agonists, carbachol and oxotremorine, but significantly increased the toxicity of another organophosphate, dichlorvos. Oral treatment of rats with CVP (15 mg/kg) inhibited brain acetylcholinesterase (AChE) activity. This inhibition became maximal at 4 hr (about 20% of control) and lasted more than 24 hr after the administration. Twenty-four hours after oral administration of CVP (15 mg/kg), the second dose (CVP 30 mg/kg, po) was less effective in inhibiting cholinesterase activities of the brain, erythrocyte, and plasma compared with naive rats treated with the same dose. The difference in brain AChE activity between control and CVP pretreatment groups was greater in magnitude than that measured in erythrocytes. CVP concentration in plasma after the oral administration of CVP (30 mg/kg) was decreased by the CVP pretreatment. Area under the concentration vs time curve (AUC) in the CVP-pretreated group was about one-fourth of AUC in the control group. This decrease in the AUC was comparable to the decrease in the toxicity of CVP. Thus, the protection against subsequent CVP challenge may be due to the reduction in the inhibition of brain AChE activity caused by the decrease in plasma CVP concentration.
Title: Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor Shibahara S, Kubo T, Perski HJ, Takahashi H, Noda M, Numa S Ref: European Journal of Biochemistry, 146:15, 1985 : PubMed
Human genomic DNA encoding the gamma subunit precursor of the skeletal muscle acetylcholine receptor has been cloned by screening a gene library with a calf cDNA probe and has been subjected to nucleotide sequence analysis. Comparison of the nucleotide sequence of the cloned human genomic DNA with that of the calf cDNA has revealed that the protein-coding sequence of this gene is divided by 11 introns into 12 exons. Evidence is presented to suggest that the human muscle acetylcholine receptor gamma and delta subunit genes are juxtaposed. The primary structure of the gamma subunit precursor of the human muscle acetylcholine receptor has been deduced from the corresponding gene sequence. This polypeptide is composed of 517 amino acids including a hydrophobic prepeptide of 22 amino acids. The gamma subunit of the human muscle acetylcholine receptor, like the alpha subunit of the same receptor as well as the alpha, beta and gamma subunits of its calf counterpart, shares structural features common to all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the alpha subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M1-M4. Thus, the human gamma subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The 12 exons seem to correspond to different structural and functional domains of the gamma subunit precursor molecule. Some exons and the protein regions encoded by them are more highly conserved between the mammalian and Torpedo sequences. The pattern of regional homology observed is consistent with the relatively high conservation of the region encompassing the putative disulphide bridge and of the region containing the putative transmembrane segments M1, M2 and M3.
Clones harbouring cDNA sequences for the beta subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has disclosed the primary structure of this polypeptide, which consists of 505 amino acids including a hydrophobic prepeptide of 24 amino acids. The beta subunit of the calf muscle acetylcholine receptor, like the alpha and gamma subunits of the same receptor and the alpha subunit of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedo beta subunits (59%) is comparable to that between the gamma subunits (56%), but is lower than that between the alpha subunits of the two species (81%). Some regions of the beta subunit molecule, including the region corresponding to the putative acetylcholine binding area on the alpha subunit and the region encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between the two species.
The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 1-3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4-6). We have recently elucidated the complete primary structures of the alpha-, beta- and delta-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides. Here, we report the whole primary structure of the gamma-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.
The nicotinic acetylcholine receptor (AChR) from fish electric organ and mammalian skeletal muscle is the best characterized neurotransmitter receptor (reviewed in refs 1-3). The AChR from the electroplax of the ray Torpedo californica consists of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 4-6); the apparent molecular weights of the alpha-, beta-, gamma- and delta-subunits are 40,000 (40K), 50K, 60K and 65K, respectively. Knowledge of the primary structures of these constituent polypeptides would facilitate the understanding of the molecular mechanism underlying the function of the neurotransmitter receptor. Recently, we have cloned cDNA for the alpha-subunit precursor of the T. californica AChR and have deduced the primary structure of this polypeptide from the nucleotide sequence of the cloned cDNA. Here we report the cloning and nucleotide analysis of cDNAs for the AChR beta- and delta-subunit precursors. The primary structures of the two polypeptides deduced from the cDNA sequences reveal conspicuous amino acid sequence homology among these and the alpha-subunits. The three subunits contain several highly conserved regions which may be essential for the receptor function or inter-subunit interaction.
DNA sequences complementary to the Torpedo californica electroplax mRNA coding for the alpha-subunit precursor of the acetylcholine receptor were cloned. The nucleotide sequence of the cloned cDNA indicates that the precursor consists of 461 amino acids including a prepeptide of 24 amino acids. Possible sites for acetylcholine binding and antigenic determinants on the alpha-subunit molecule are discussed.
