Title: Analysis of Adverse Events of Cholinesterase Inhibitors and NMDA Receptor Antagonists on Arrhythmias Using the Japanese Adverse Drug Event Report Database Kobayashi S, Sugama N, Nagano H, Miyamori A, Takahashi M, Kushiyama A Ref: Drugs Real World Outcomes, :, 2023 : PubMed
BACKGROUND: The association between anti-dementia drugs and arrhythmia is uncertain. In addition, the effects of certain drug combinations are not yet well known. OBJECTIVE: We investigated the association between anti-dementia drugs and arrhythmia. Furthermore, we investigated the effects of anti-dementia drugs both alone and in combination on the likelihood of arrhythmia in patients with dementia. METHODS: We examined the Japanese Adverse Drug Event Report database (JADER) from April 2004 to May 2022 for dementia drug users aged <= 60 years. We calculated the unadjusted reported odds ratio (ROR) and adjusted ROR for confounding factors. Furthermore, we examined the association of various combinations of anti-dementia drugs with the development of arrhythmias. RESULTS: There were 6718 arrhythmia cases identified out of 333,702 reported cases. The unadjusted ROR results were as follows: donepezil alone (ROR 4.39, 95% confidence interval [CI] 3.89-4.95), rivastigmine alone (2.10, 1.53-2.87), galantamine alone (3.87, 3.04-4.94), memantine alone (2.25, 1.59-3.20), and combination of choline esterase inhibitor and memantine (2.56, 1.84-3.57). In a multivariate analysis, the RORs remained significant. CONCLUSIONS: Regardless of whether anti-dementia drugs were used alone or in combination, attention should be paid to the occurrence of arrhythmias.
Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1-150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.
Although the effects of prenatal passive smoking on birth weight have been reported, the effects of metabolic gene polymorphisms on passive smoking have not been studied. Therefore, we investigated the effects of maternal passive smoking and metabolic gene polymorphisms on child growth up to 3years of age using cotinine as a biomarker. We included 1356 Japanese participants in a prospective cohort between 2003 and 2007 (cotinine levels at the third trimester<=0.21ng/mL and 0.22 to 11.48ng/mL for non-passive and passive smokers, respectively), and measured child outcomes such as weight, length, head circumference, and Kaup index. Additionally, we analyzed cytochrome P450 1A1 (CYP1A1), epoxide hydrolase 1 (EPHX1), and two N-acetyltransferase 2 (NAT2) genotypes using real-time polymerase chain reaction methods. Associations were investigated using multiple regression models. Kaup index gain from birth up to 3years of age was significantly smaller in children born to passive smokers than in those born to non-passive smokers (-0.34kg/m2; 95% confidence interval: -0.67, -0.01). Maternal CYP1A1 genotype was not associated with prenatal passive smoking and Kaup index gain, but was significantly associated with prenatal passive smoking and head circumference gain from birth up to 3years of age (-0.75cm; 95% confidence interval: -1.39, -0.12). Thus, this study suggests that prenatal passive smoking may have potent effects on postnatal growth from birth up to 3years of age. Moreover, children with maternal CYP1A1 genotype may be more susceptible to the effects of prenatal passive smoking.
Acetylcholine plays various important roles in the central nervous system of invertebrates as well as vertebrates. In the olfactory center of the terrestrial slug Limax, the local field potential (LFP) oscillates, and the change in its oscillatory frequency is thought to correlate with the detection of odor that potentially changes an ongoing behavior of the animal. Acetylcholine is known to upregulate the frequency of the LFP oscillation, and is one of the candidates for the neurotransmitters that are involved in such higher cognitive functions. However, there have been no histological data on the cholinergic system in gastropods, nor are there data on the receptors that are responsible for the upregulation of the oscillatory frequency of LFP due to the lack of analytical tools (such as antibodies or cDNA sequence information on cholinergic system-related genes). Here we cloned the cDNAs of choline acetyltransferase (ChAT), acetylcholinesterase, vesicular acetylcholine transporter, and several nicotinic acetylcholine receptors (nAChRs), and investigated their localization in the brain of Limax. We also generated a polyclonal antibody against ChAT to examine its localization, and investigated pharmacologically the involvement of nAChRs in the LFP oscillation. Our data showed: 1) dense distribution of the neurons expressing mRNAs of ChAT and vesicular acetylcholine transporter in the olfactory center; 2) spatially unique expression patterns of different nAChRs in the olfactory center; 3) involvement of nAChRs in the upregulation of the oscillation; 4) localization of ChAT protein in nerve fibers and/or terminals; and 5) the presence of cholinergic nerves in the tentacles. J. Comp. Neurol. 522:2951-2966, 2014. (c) 2014 Wiley Periodicals, Inc.
We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 mus time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or alpha-bungarotoxin (alphaBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.
n-Butyl D- and L-lactates (BuDLa and BuLLa) were incubated with immobilized lipase. (1)H-NMR showed that BuDLa reacted to oligomers, while BuLLa did not react. A mixture containing 90.4% of BuLLa and 9.6% of BuDLa was incubated with the enzyme for 72 h, then distilled. The purity of BuLLa increased to 98.6%.
Title: Lipase-catalyzed polyester synthesis--a green polymer chemistry Kobayashi S Ref: Proc Jpn Acad Ser B Phys Biol Sci, 86:338, 2010 : PubMed
This article is a short comprehensive review describing in vitro polyester synthesis catalyzed by a hydrolysis enzyme of lipase, most of which has been developed for these two decades. Polyesters are prepared by repeated ester bond-formation reactions; they include two major modes, ring-opening polymerization (ROP) of cyclic monomers such as cyclic esters (lactones) and condensation polymerization via the reaction between a carboxylic acid or its ester group and an alcohol group. Polyester synthesis is, therefore, a reaction in reverse way of in vivo lipase catalysis of ester bond-cleavage with hydrolysis. The lipase-catalyzed polymerizations show very high chemo-, regio-, and enantio-selectivities and involve various advantageous characteristics. Lipase is robust and compatible with other chemical catalysts, which allows novel chemoenzymatic processes. New syntheses of a variety of functional polyesters and a plausible reaction mechanism of lipase catalysis are mentioned. The polymerization characteristics are of green nature currently demanded for sustainable society, and hence, desirable for conducting 'green polymer chemistry'.
Title: Lipase-catalyzed oligomerization and hydrolysis of alkyl lactates: direct evidence in the catalysis mechanism that enantioselection is governed by a deacylation step Ohara H, Onogi A, Yamamoto M, Kobayashi S Ref: Biomacromolecules, 11:2008, 2010 : PubMed
Lipase-catalyzed oligomerization of alkyl d- and l-lactate monomers (RDLa and RLLa, respectively) was studied for the first time. It has been found that the oligomerization occurs enantioselectively only for d-lactates to give oligomers up to heptamers of lactic acid (LA) in good to high yields by using primary C1 to C8 alkyl groups and sec-butyl group for d-lactate monomers. No reaction happened for all l-lactates in similar conditions. Lipase-catalyzed hydrolysis of alkyl d- and l-lactates was also examined, revealing that the hydrolysis took place for both d- and l-lactates, although l-lactates proceeded a couple of times slower. The hydrolysis results clearly demonstrate that the lipase catalysis mechanism involves an acyl-enzyme intermediate (EM) formation via the acylation step from both d- and l-lactates as a rate-determining step, and the subsequent deacylation step, a nucleophilic attack of water to the EM, takes place to produce free LA. On the other hand, in the oligomerization of d-lactates, the deacylation step, in which a sec-alcohol group of the monomer or of the propagating chain-end attacks to the EM, is only allowed for the sec-d-alcohol group to give a one-LA-unit-elongated oligomer. l-Lactates form the EM; however, the subsequent deacylation reaction with both the sec-l- and sec-d-alcohol groups does not take place, failing in the oligomerization to occur. These results provide with the first direct evidence in the lipase catalysis that the enantioselection is governed by the deacylation step. In the co-oligomerization between l- and d-lactates, the l-isomer retarded the reaction rate of the d-isomer, which was found due to the function of the former as a competitive inhibitor in the acylation step toward the latter.
Title: Recent developments in lipase-catalyzed synthesis of polyesters Kobayashi S Ref: Macromol Rapid Commun, 30:237, 2009 : PubMed
Polyester synthesis by lipase catalyst involves two major polymerization modes: i) ring-opening polymerization of lactones, and, ii) polycondensation. Ring-opening polymerization includes the finding of lipase catalyst; scope of reactions; polymerization mechanism; ring-opening polymerization reactivity of lactones; enantio-, chemo- and regio-selective polymerizations; and, chemoenzymatic polymerizations. Polycondensation includes polymerizations involving condensation reactions between carboxylic acid and alcohol functional groups to form an ester bond. In most cases, a carboxylic acid group is activated as an ester form, such as a vinyl ester. Many recently developed polymerizations demonstrate lipase catalysis specific to enzymatic polymerization and appear very useful. Also, since lipase-catalyzed polyester synthesis provides a good opportunity for conducting "green polymer chemistry", the importance of this is described.
Title: Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens Makioka A, Kumagai M, Kobayashi S, Takeuchi T Ref: Parasitol Res, 105:977, 2009 : PubMed
Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.
Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.
Title: Quantitative analysis of the effects of donepezil on regional cerebral blood flow in Alzheimer's disease by using an automated program, 3DSRT Tateno M, Kobayashi S, Utsumi K, Morii H, Fujii K Ref: Neuroradiology, 50:723, 2008 : PubMed
INTRODUCTION: Donepezil, an acetylcholinesterase inhibitor, has been reported to have an effect that improves cerebral blood flow (CBF) alongside its primary effect on memory function. The aim of this study was to investigate the effects of long-term, low-dose donepezil therapy on blood perfusion in Alzheimer's disease (AD) by using a fully automated regional CBF quantification program named 3DSRT. MATERIALS AND METHODS: Fifteen subjects with mild to moderate AD according to NINCDS/ADRDA criteria underwent 99mTc-ethylcysteinate dimer (ECD) brain perfusion single photon emission computed tomography (SPECT) twice with an interval of 55.1 +/- 11.0 weeks. The dose of donepezil was fixed at 5 mg/day following the induction period (3 mg/day) of 2 weeks. Clinical efficacy of donepezil was assessed by using the Mini-Mental State Examination (MMSE). The results of SPECT imaging under exactly identical conditions were analyzed by 3DSRT, which enables us to perform a very objective assessment. RESULTS: Despite a decrease of the MMSE score from 20.9 +/- 4.7 to 18.7 +/- 5.7, CBF was increased in almost all cerebral areas except the left temporal segment. The increase was statistically significant in the left callosomarginal, right central, and bilateral pericallosal and lenticular nucleus segments. CONCLUSION: Thus far, no direct cerebrovascular effects have been reported for donepezil. We hypothesize that these CBF-promoting effects of donepezil might be related to increased neuronal activity and enhanced connection of neurons.
Title: Comprehensive evaluation of genetic and environmental factors influencing the plasma lipoprotein-associated phospholipase A2 activity in a Japanese population Zhang SY, Shibata H, Karino K, Wang BY, Kobayashi S, Masuda J, Nabika T Ref: Hypertens Res, 30:403, 2007 : PubMed
The lipoprotein-associated phospholipase A2 (Lp-PLA2) metabolizes oxidized phospholipids, generating lysophosphatidylcholine. The activity of the enzyme is known to be influenced largely by a single-nucleotide polymorphism, G994T, in the Lp-PLA2 gene. Interestingly, this polymorphism is much more prevalent in Japanese than Caucasians. The purpose of the current study was to evaluate the effects of the G994T, several environmental factors, and their interactions on the Lp-PLA2 activity in a large Japanese cohort. Participants (1,110 males and 908 females) of a health-screening examination were recruited for this study. Genotyping of the G994T was done using allele-specific polymerase chain reaction (PCR). The Lp-PLA2 activity was measured using commercial kits. The minor allele (994T) frequency of the polymorphism was 0.17 in this study, which was consistent with previous reports. According to the multivariate linear regression analysis, the G994T was the most potent factor influencing the enzyme activity (standardized beta=0.76), followed by the low-density lipoprotein cholesterol (LDL-C) level (standardized beta=0.32) and the sex (standardized beta=0.13). The LDL-C level showed a significant interaction with the G994T genotype. By contrast, no significant interaction was observed between the LDL-C level and the sex. These observations should provide useful information for future clinical and epidemiological evaluations of the Lp-PLA2 activity in cardiovascular diseases in Japanese.
Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.
Nicotinic acetylcholine receptors are key molecules in cholinergic transmission in the nervous system. Because of their structural complexity, only a limited number of subtype-specific agonists and antagonists are available to study nicotinic receptor functions. To overcome this limitation, we used voltageclamp recordings to examine the effects of several frog skin alkaloids on acetylcholine-elicited currents in Xenopus laevis oocytes expressing major types of neuronal nicotinic receptors (alpha4beta2, alpha7, alpha3beta2, alpha3beta4, and alpha4beta4). We found that the 5,8-disubstituted indolizidine (-)-235B' acted as a potent noncompetitive blocker of alpha4beta2 nicotinic receptors (IC50 = 74 nM). This effect was highly selective for alpha4beta2 receptors compared with alpha3beta2, alpha3beta4, and alpha4beta4 receptors. The inhibition of alpha4beta2 currents by (-)-235B' was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 microM). Moreover, the blockade of alpha4beta2 currents by (-)-235B' was voltage-dependent (more pronounced at hyperpolarized potentials) and use-dependent, indicating that (-)-235B' behaves as an open-channel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((-)-223A and (+)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((+)-207I) were less potent than (-)-235B', and none showed selectivity for alpha4beta2 receptors. The quinolizidine (-)-1-epi-207I and the tricyclic (+)-205B had 8.7- and 5.4-fold higher sensitivity, respectively, for inhibition of the alpha7 nicotinic receptor than for inhibition of the alpha4beta2 receptor. These results show that frog alkaloids alter the function of nicotinic receptors in a subtype-selective manner, suggesting that an analysis of these alkaloids may aid in the development of selective drugs to alter nicotinic cholinergic functions.
Our previous work identified a genetic mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/Snk mice (previously KK/San), a mutant strain of KK obese mice. KK/Snk had significantly lower plasma triglyceride and free fatty acid (FFA) than KK mice. Human ANGPTL3 treatment increased both plasma triglyceride and FFA. ANGPTL3 inhibited the activity of lipoprotein lipase, which accounted for the increase of plasma triglyceride. The mechanism how ANGPTL3 affects plasma FFA has not been known. The current study reveals that ANGPTL3 targets on adipose cells and induces lipolysis. Both plasma FFA and glycerol decreased in KK/Snk and increased by the treatment of human ANGPTL3. Specific bindings of ANGPTL3 to adipose cells were shown using fluorescence-labeled protein visually and 125I-labeled protein by the binding analysis. Furthermore, ANGPTL3 activated the lipolysis to stimulate the release of FFA and glycerol from adipocytes. We conclude that ANGPTL3 is a liver-derived lipolytic factor targeting on adipocyte.
Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.
Title: Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects Namekawa S, Suda S, Uyama H, Kobayashi S Ref: Int J Biol Macromol, 25:145, 1999 : PubMed
Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.
The ternary allosteric model predicts the possibility of discovering molecules with novel and highly subtype-selective modes of action. This approach has been applied to muscarinic receptors. The alkaloid brucine is capable of selectively enhancing by an allosteric mechanism the effects of low but not high concentrations of acetylcholine at only the m1 subtype of muscarinic receptors. A simple derivative of brucine, N-chloromethylbrucine, enhances acetylcholine actions selectively at only m3 receptors. In addition it binds to, but does not affect, the properties of m4 receptors, thereby demonstrating neutral cooperativity and an 'absolute' selectivity of action at m3 receptors over m4 receptors. Brucine N-oxide enhances acetylcholine binding at m3 and m4 receptors and is neutral at m1 and m5 receptors. These findings allow the possibility of developing muscarinic agents that have a novel and highly targeted mode of action; they may act only on a single muscarinic receptor subtype which is functioning sub-optimally and therefore be of use therapeutically in the early stages of Alzheimer's Disease.
The mouse Peg1/Mest gene is an imprinted gene that is expressed particularly in mesodermal tissues in early embryonic stages. It was the most abundant imprinted gene among eight paternally expressed genes (Peg 1-8) isolated by a subtraction-hybridization method from a mouse embryonal cDNA library. It has been mapped to proximal mouse chromosome 6, maternal duplication of which causes early embryonic lethality. The human chromosomal region that shares syntenic homology with this is 7q21-qter, and human maternal uniparental disomy 7 (UPD 7) causes apparent growth deficiency and slight morphological abnormalities. Therefore, at least one paternally expressed imprinted gene seems to be present in this region. In this report, we demonstrate that human PEG1/MEST is an imprinted gene expressed from a paternal allele and located on chromosome 7q31-34, near D7S649. It is the first imprinted gene mapped to human chromosome 7 and a candidate for a gene responsible for primordial growth retardation including Silver-Russell syndrome (SRS).
Title: Possible nicotinic receptor-mediated modulation of synaptic transmission in nucleus of the solitary tract Shiraki T, Toyoda A, Sugino H, Hori A, Kobayashi S Ref: American Journal of Physiology, 272:R869, 1997 : PubMed
Signal transmission from afferent nerves to neurons in the nucleus of the solitary tract (NTS) may be mediated partially by nicotinic acetylcholine receptors (nAChRs). Here, we investigated nAChR-mediated signal transmission using rat NTS slices. First, we characterized nAChRs by obtaining patch-clamp recordings from NTS neuronal cell bodies. Under whole cell voltage-clamp conditions at -60 mV, application of nicotine induced an inward current, and this effect was blocked by hexamethonium. In outside-out patch recordings, nicotine was seen to induce a hexamethonium-sensitive single-channel current. Second, we investigated nAChR-mediated signal transmission. Fast synaptic transmission mediated by nAChRs was not detected. The action of diffusible acetylcholine (ACh) on nAChRs was then tested using the outside-out patches excised from NTS neurons as probes for ACh. When the patch was placed at a distance of 20-30 microm from the cell body, single-channel currents were recorded, and these were inhibited by hexamethonium. The frequency of channel opening was increased by high-extracellular potassium concentration solution suggesting the voltage-dependent release ofACh that acts on nAChRs. These results suggested that nAChR-mediated signal transmission from sensory afferents to NTS neurons is in part mediated by diffusible ACh.
Title: Impaired acquisition in a 14-unit T-maze following medial septal lesions in rats is correlated with lesion size and hippocampal acetylcholinesterase staining Kametani H, Spangler EL, Bresnahan EL, Kobayashi S, Long JM, Ingram DK Ref: Physiology & Behavior, 53:221, 1993 : PubMed
Septohippocampal cholinergic system involvement in acquisition of an aversively motivated 14-unit T-maze was evaluated in 4-month-old male Fischer-344 rats. Each rat was assigned to one of two groups that received either a bilateral electrolytic lesion to the medial septal area (MSA) or a sham operation. One week after surgery, each rat began pretraining in one-way active avoidance (footshock = 0.8 mA) consisting of 10 trials per day on each of 3 consecutive days. Criterion for successful completion of pretraining was 8/10 avoidances on the third day. On the day following completion of pretraining, each rat received 10 trials in a shock-motivated 14-unit T-maze. The performance requirement was to move through each of five maze segments within 10 s to avoid footshock (0.8 mA). A second 10-trial session was provided 24 h later. Performance measures included errors, alternation errors, runtime, shock frequency, and duration. Following maze training, each rat was sacrificed, and formalin-fixed brains were frozen for histology, which included procedures for thionin Nissl and acetylcholinesterase (AChE) staining. MSA-lesioned rats were observed to be significantly impaired on all measures of maze performance compared to sham-operated controls. Densitometric analysis of hippocampal AChE staining revealed a 30% reduction in relative AChE staining of MSA-lesioned rats compared to sham-operated controls. Lesion size was observed to be highly positively correlated with maze errors. A negative correlation of mean error score with density of AChE staining was observed for MSA-lesioned rats, but not for sham-operated rats.
Title: Enzymatic Ring-Opening Polymerization of Lactones Catalyzed by Lipase Uyama H, Kobayashi S Ref: Chem Lett, 22:1149, 1993 : PubMed
Enzymatic ring-opening polymerization of lactones was achieved by using lipase as catalyst. The polymerization of epsilon-caprolactone by Pseudomonas fluorescens lipase at 60 C in bulk for 10 days afforded a polyester with average molecular weight of 7.0 x 103. From 1H and 13C NMR analysis, the polymer possesses the terminal structure of a carboxylic acid group at one end and a hydroxyl group at the other.
