OBJECTIVES: Acetylcholinesterase (AChE) staining has become the gold standard for definitively diagnosing Hirschsprung disease (HD), although some pitfalls have been reported. We reevaluated a large series at our institute in order to validate the accuracy of AChE staining for detecting HD. METHODS: A retrospective study of the rectal mucosal specimens of all of the children with suspected HD during a 13-year period was performed. The specimens were stained according to the modified Karnovsky-Roots method for AChE staining. The final diagnosis, prognosis, and management after the histopathological diagnosis were analyzed with a questionnaire sent to the patient's original hospital. RESULTS: Three hundred and fifty-eight specimens were collected. One hundred twenty-two (34%) specimens were diagnosed as HD, 198 (55%) as nonHD, 25 (7%) as "undetermined," and 13 (4%) as "inappropriate." The non-HD group contained 190 (96%) specimens with a normal appearance and 8 (4%) specimens with suspected intestinal neuronal dysplasia (IND). Three hundred and six of 358 questionnaires were returned. The final diagnosis showed that no specimens first diagnosed as HD were identified as non-HD and vice versa, for a sensitivity and specificity of 100%. Four cases were finally diagnosed as chronic idiopathic intestinal pseudo-obstruction (CIIP) in the non-HD group. All of the patients with HD underwent radical surgery. Most non-HD patients were managed conservatively, although some continued to have constipation. CONCLUSIONS: AChE staining is an accurate tool for differentiating between HD and non-HD with high sensitivity and specificity. CIIP can be included in cases of non-HD; therefore, careful follow-up is mandatory.
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.
The enzymes LinBUT and LinBMI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of beta-hexachlorocyclohexane (beta-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinBMI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinBMI were categorized into three groups based on the efficiency of the first-step (from beta-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinBMI and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinBMI. The dynamics simulation analyses of wild-type LinBMI and LinBUT revealed that the entrance of the substrate access tunnel of LinBUT was more flexible than that of LinBMI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.
BACKGROUND: The inhibition of dipeptidyl peptidase-4 (DPP4) protects the heart from acute myocardial ischemia. However, the role of DPP4 in chronic heart failure independent of coronary artery disease remains unclear. METHODS AND RESULTS: We first localized the membrane-bound form of DPP4 to the capillary endothelia of rat and human heart tissue. Diabetes mellitus promoted the activation of the membrane-bound form of DPP4, leading to reduced myocardial stromal cell-derived factor-1alpha concentrations and resultant angiogenic impairment in rats. The diabetic rats exhibited diastolic left ventricular dysfunction (DHF) with enhanced interstitial fibrosis caused partly by the increased ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 in a DPP4-dependent fashion. Both genetic and pharmacological DPP4 suppression reversed the stromal cell-derived factor-1alpha-dependent microvasculopathy and DHF associated with diabetes mellitus. Pressure overload induced DHF, which was reversed by DPP4 inhibition via a glucagon-like peptide-1/cAMP-dependent mechanism distinct from that for diabetic heart. In patients with DHF, the circulating DPP4 activity in peripheral veins was associated with that in coronary sinus and with E/e', an echocardiographic parameter representing DHF. Comorbid diabetes mellitus increased the circulating DPP4 activities in both peripheral veins and coronary sinus. CONCLUSIONS: DPP4 inhibition reverses DHF via membrane-bound DPP4/stromal cell-derived factor-1alpha-dependent local actions on angiogenesis and circulating DPP4/glucagon-like peptide-1-mediated inotropic actions. Myocardium-derived DPP4 activity in coronary sinus can be monitored by peripheral vein sampling, which partly correlates with DHF index; thus, circulating DPP4 may potentially serve as a biomarker for monitoring DHF.
Thermus thermophilus HB8, an extremely thermophilic bacterium. The open reading frame of TTHA1809 from Thermus thermophilus HB8 was annotated as a proline iminopeptidase-related protein. Proline iminopeptidase (PIP) is a serine peptidase that catalyzes the removal of N-terminal proline from peptides with high specificity. In this study, we report the crystal structure of the proline iminopeptidase-related protein TTHA1809 from Thermus thermophilus HB8, and compare the active site of the tricorn-interacting aminopeptidase F1, which is the best homolog found using the Dali program, with the corresponding region of TTHA1809. Comparison with the tricorn-interacting aminopeptidase F1: The tricorn-interacting aminopeptidase F120 from Thermoplasma acidophilum has a Ser-His-Asp catalytic triad in the active site. The superimposition between TTHA1809 and F1 revealed that the residue corresponding to the catalytic Ser105 in F1 is replaced by a Gly at TTHA1809. Asp229 and His255 of TTHA1809 were located at the same position as Asp244 and His271 of the catalytic triad of F1. His255 of TTHA1809 was located in a loop between the beta8-strand and the alpha10-helix. The ND-1 atom of His255 was hydrogen-bonded to Asp229 in a loop located between the beta7-strand and the alpha9-helix, whereas the NE-2 atom of His255 formed a hydrogen bond with a water molecule because of the lack of catalytic Ser residue. Thus, TTHA1809 and F1 would have different functions. A BLAST search using TTHA1809 revealed the possibility of the existence of enzymes lacking a catalytic Ser residue in several microorganisms (Thermus thermophilus HB27, Deinococcus geothermalis DSM 11300, Legionella pneumophila subsp. pneumophila str. Philadelphia 1, Dechloromonas aromatica RCB, and Hahella chejuensis KCTC 2396), but their functions have not yet been revealed.
Title: Detection of a new N-oxidized metabolite of flutamide, N-[4-nitro-3-(trifluoromethyl)phenyl]hydroxylamine, in human liver microsomes and urine of prostate cancer patients Goda R, Nagai D, Akiyama Y, Nishikawa K, Ikemoto I, Aizawa Y, Nagata K, Yamazoe Y Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 34:828, 2006 : PubMed
Flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide), a nonsteroidal antiandrogen, is used in the treatment of prostate cancer but is occasionally associated with hepatic dysfunction. In the present study, the metabolism of flutamide including the formation of the possible reactive toxic metabolites was investigated using human liver microsomes and 10 isoforms of recombinant human cytochrome P450 (P450). 2-Hydroxyflutamide (OH-flutamide) and 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1) were the main products of flutamide metabolism in human liver microsomes. The formation of OH-flutamide was markedly inhibited by ellipticine, an inhibitor of CYP1A1/1A2, and was mainly catalyzed by the recombinant CYP1A2. FLU-1 was also produced from OH-flutamide, but its metabolic rate was much less than that from flutamide. An inhibitor of carboxylesterase, bis-(p-nitrophenyl)phosphoric acid, completely inhibited the formation of FLU-1 from flutamide in human liver microsomes. A new metabolite, N-[4-nitro-3-(trifluoromethyl)phenyl]hydroxylamine (FLU-1-N-OH), was detected as a product of the reaction of FLU-1 with human liver microsomes and identified by comparison with the synthetic standard. The formation of FLU-1-N-OH was markedly inhibited by the addition of miconazole, an inhibitor of CYP3A4, and was mediated by recombinant CYP3A4. Furthermore, FLU-1-N-OH was detected mostly as the conjugates (glucuronide/sulfate) in the urine of prostate cancer patients collected for 3 h after treatment with flutamide. The formation of FLU-1-N-OH, however, did not differ between patients with and without abnormalities of hepatic functions among a total of 29 patients. The lack of an apparent association of the urinary excretion of FLU-1-N-OH and hepatic disorder may suggest the involvement of an additional unknown factor in the mechanisms of flutamide hepatotoxicity.
Title: Single-channel analyses of ethanol modulation of neuronal nicotinic acetylcholine receptors Zuo Y, Nagata K, Yeh JZ, Narahashi T Ref: Alcohol Clin Exp Res, 28:688, 2004 : PubMed
BACKGROUND: We have previously reported that ethanol potentiates the acetylcholine-induced currents of the alpha4beta2 neuronal nicotinic acetylcholine receptors in rat cortical neurons and of those that are stably expressed in human embryonic kidney cells. The potentiation of the maximal currents evoked by high concentrations of acetylcholine suggests that ethanol affects the channel gating. METHODS: We performed single-channel patch-clamp experiments to elucidate the detailed mechanism of ethanol modulation of the alpha4beta2 receptor that is stably expressed in human embryonic kidney cells. RESULTS: At least two conductance states, 40.5 pS and 21.9 pS, were activated by acetylcholine. Acetylcholine at 30 nM predominantly induced the high conductance state currents (85% of total). Ethanol did not affect the single-channel conductance but selectively modulated the high-conductance state currents. The high-conductance state currents exhibited two open time constants. Both time constants were increased by 100 mM ethanol, from 1.9 msec to 2.8 msec and from 9.0 msec to 15.5 msec, respectively. Ethanol also prolonged the burst duration and the open time within burst and increased the probability of channel opening. CONCLUSIONS: These changes in single-channel parameters indicate that ethanol stabilizes the alpha4beta2 receptor-channel in the opening state, explaining how the maximum acetylcholine-induced whole-cell currents are further potentiated by ethanol.
Renal microsomal cytochrome P-450 monooxygenase-dependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension.
(1) Modulation of the function of the GABA(A) and neuronal nicotinic acetylcholine receptor channels caused by general anesthetics and modulation of the GABA(A) receptor-channel by halothane, enflurane, isoflurane, and n-octanol was channel state-dependent. (3) Halothane modulation of the GABA(A) receptor was independent of subunits, but n-octanol modulation was subunit-dependent. (4) Ethanol at 30-100 microM was very potent in accelerating the desensitization of currents induced by acetylcholine. (5) The ethanol modulation was subunit- and state-dependent, occurring in the alpha3beta4 combination but only weakly in the alpha3beta2 combination. (6) In contrast, halothane at 430 microM (approximately 1 MAC) potently suppressed ACh-induced currents in the alpha3beta2 subunit combination.
Title: Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels Nagata K, Huang CS, Song JH, Narahashi T Ref: Brain Research, 769:211, 1997 : PubMed
Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 microM carbachol was enhanced by the first co-application with 10 or 100 microM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 microM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3-3 microM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.
Title: Isolation and characterization of human liver cytochrome b5 cDNA Miyata M, Nagata K, Yamazoe Y, Kato R Ref: Pharmacol Res, 21:513, 1989 : PubMed
Three cDNA clones encoding human cytochrome b5 have been isolated and sequenced from a lambda gt 11 cDNA library of a human liver. The largest clone (b5-C) contains 765 base pairs corresponding to a complete coding sequence of 134 amino acids and sequences for 5' non-coding (56 bases) and 3' non-coding regions (307 bases). In Northern blots, a band hybridizing to 32P-labelled b5-B cDNA clone was detected at around 12S in mRNA of human and rat livers. The deduced amino acid sequence was identical with the partial amino acid sequence (2-100th) previously reported, except that two amino acid replacements were observed between the 89th and 91st positions. The deduced amino acid sequence of human cytochrome b5 shares 88.0, 86.5, 86.5 and 74.4% similarity with those of rat, pig, horse and chicken, respectively. The sequences in the haem binding region were highly conserved among these species. In addition, high similarities in the hydrophobicity profiles were maintained throughout their entire regions, although some diversity was observed in the N-terminal sequences between human and chicken.
