Title: Non-linear model analysis of the relationship between cholinesterase activity in rats exposed to 2, 2-dichlorovinyl dimethylphosphate (dichlorvos) and its metabolite concentrations in urine Sato H, Ito Y, Hanai C, Nishimura M, Ueyama J, Kamijima M Ref: Toxicology, :152679, 2021 : PubMed
Urinary dialkylphosphates (DAPs) are measured to assess exposure to organophosphorus pesticides (OPs), but they are common metabolites of OPs and not specific indices for individual agents. Biomonitoring (BM) of urinary DAPs has been widely adopted as an assessment of individual exposure in general environments, however, guidance values for DAPs based on health effects have yet to be established. The present study aimed to clarify the relationship between the amount of urinary dimethylphosphate (DMP), a metabolite of dichlorvos (DDVP), and the inhibition of cholinesterase (ChE) activity in rats exposed to DDVP. The relationship was analyzed using a nonlinear model analysis, and the excretion level of urinary DMP equivalent to ChE 20% inhibition (EL20) and the lower limit of the 95% confidence interval of EL20 (ELL20) were estimated. EL20 and ELL20 (mg/24 h urine) of brain, erythrocyte, and plasma ChE activities after 10-day administration of DDVP were 0.21 and 0.15, 0.11 and 0.06, and 0.23 and 0.09, respectively. Extrapolating ELL20 of the brain ChE to humans, the range of 24 h urinary DMP concentration according to the 20% inhibition of cholinesterase activity was estimated to be 20.5-30.8 mg/l. In conclusion, the amount of urinary DMP as ELL20 for DDVP exposure was identified and could probably be used as a novel index for the assessment of risk from OP exposure. Further studies are needed to clarify the ELL20 s derived from OPs other than DDVP, for informing efforts to establish guidance values of urinary OP metabolites that should prevent neurotoxicity.
We gave mice a 540 mg/kg dose of LD50 acephate, followed by an assessment of acephate, methamidophos (MP), and choline esterase (ChE) activity for up to 4 hours (hr) in order to investigate the time course of acephate intoxication. At 1 hr, the blood acephate and MP levels were 428 +/- 90 microg/ml (mean +/- SEM) and 4.2 +/- 0.4 microg/ ml, respectively. The liver acephate levels were similar to those in the blood, but the liver MP levels were approximately 3.5 times that of the blood at 1 hr. The brain MP level tended to be higher than the blood MP at 1 hr. These levels decreased gradually over 4 hr, but the brain acephate and MP levels surpassed the blood levels significantly at 4 hr, and after 2 hr, respectively. Serum, liver, cerebrum, cerebellum, and brainstem cholinesterase activity (ChE) were inhibited at 1 hr, and remained inhibited in all but the cerebellum until the end of the experiment. The obtained data were applied to previously reported autopsy cases of acephate intake. Experimental data suggest that brain MP is involved in acute acephate-induced poisoning, even after a reduction in blood acephate. In autopsy cases with suspected acephate poisoning, the MP level in the brain should be considered in addition to the ChE activity to diagnose the cause of death.
Middle East respiratory syndrome coronavirus (MERS-CoV) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. Several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. Here, we developed transgenic (Tg) mice on a C57BL/6 background; these mice expressed human CD26/dipeptidyl peptidase 4 (hDPP4), a functional receptor for MERS-CoV, under the control of an endogenous hDPP4 promoter. We then characterized this mouse model of MERS-CoV. The expression profile of hDPP4 in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hDPP4 was overexpressed in murine CD3-positive cells within peripheral blood and lymphoid tissues. Intranasal inoculation of young and adult Tg mice with MERS-CoV led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within 7 days, with only transient loss of body weight. However, the immunopathology in young and adult Tg mice was different. On day 5 or 7 postinoculation, lungs of adult Tg mice contained higher levels of proinflammatory cytokines and chemokines associated with migration of macrophages. These results suggest that the immunopathology of MERS-CoV infection in the Tg mouse is age dependent. The mouse model described here will increase our understanding of disease pathogenesis and host mediators that protect against MERS-CoV infection.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) infections are endemic in the Middle East and a threat to public health worldwide. Rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of MERS-CoV infection based on transgenic mice expressing human DPP4 (hDPP4). The pattern of hDPP4 expression in this model was similar to that in human tissues (except lymphoid tissue). In addition, MERS-CoV was limited to the respiratory tract. Here, we focused on host factors involved in immunopathology in MERS-CoV infection and clarified differences in antiviral immune responses between young and adult transgenic mice. This new small-animal model could contribute to more in-depth study of the pathology of MERS-CoV infection and aid development of suitable treatments.
        
Title: Contribution of intestinal dipeptidyl peptidase-4 inhibition for incretin-dependent improved glucose tolerance in mice Yamashita S, Kawakami Y, Sato H, Sugitani S, Goto M, Kato N Ref: European Journal of Pharmacology, :172521, 2019 : PubMed
Dipeptidyl peptidase-4 (DPP-4) inhibitors prevent the degradation of glucagon-like peptide-1 (GLP-1) and improve glycemic control. The GLP-1 insulinotropic effect involves a pathway through vagus nerve GLP-1 receptors in the gut, in addition to a direct effect on the pancreas. Therefore, this study verified whether DPP-4 inhibition in the gut contributed to the improvement of glycemic control. Anagliptin, a DPP-4 inhibitor, was administered orally or subcutaneously (with or without passing through the gastrointestinal tract, respectively) to mice. The association between blood glucose suppression following oral glucose challenge and DPP-4 inhibition in the small intestine and plasma was assessed. Oral administration of anagliptin (0.03-0.3mg/kg) in normal mice significantly suppressed blood glucose, which was associated with an increase in insulin secretion at a dose of >/=0.1mg/kg (P<0.05). Subcutaneous administration of anagliptin (0.01-0.1mg/kg) produced similar results. However, plasma DPP-4 inhibition following oral administration was weaker than that following subcutaneous administration; blood glucose suppression was significantly correlated with small intestinal DPP-4 inhibition (r=0.949, P<0.01), but not with plasma DPP-4 inhibition. Additionally, similar results were observed in a type 2 diabetes model (r=0.975, P<0.001). Thus, these results demonstrated that an improvement in glycemic control was dependent upon small intestinal DPP-4 inhibition. As these effects were accompanied by the elevation of intact GLP-1 in the portal, this suggests that improvement in glucose tolerance after anagliptin treatment might be related to an increase in GLP-1 receptor signaling in the small intestine and portal vein.
The regioisomers of the di- and mono-oleate of monochloropropanediol (MCPD) have been synthesized and subsequently hydrolyzed with pancreatic lipase and pancreatin to estimate the intestinal digestion and absorption of these compounds after their intake. The hydrolysates were analyzed by HPLC using a corona charged aerosol detection system, which allowed for the separation and detection of the different regioisomers of the MCPD esters. The hydrolysates were also analyzed by GC-MS to monitor the free MCPD. The results indicated that the two acyl groups of 2-MCPD-1,3-dioleate were smoothly hydrolyzed by pancreatic lipase and pancreatin to give free 2-MCPD. In contrast, the hydrolysis of 3-MCPD-1,2-dioleate proceeded predominantly at the primary position to produce 3-MCPD-2-oleate. 2-MCPD-1-oleate and 3-MCPD-1-oleate were further hydrolyzed to free 2- and 3-MCPD by pancreatic lipase and pancreatin, although the hydrolysis of 3-MCPD-2-oleate was 80 % slower than that of 3-MCPD-1-oleate. The intestinal absorption characteristics of these compounds were evaluated in vitro using a Caco-2 cell monolayer. The results revealed that the MCPD monooleates, but not the MCPD dioleates, were hydrolyzed to produce the free MCPD in the presence of the Caco-2 cells. The resulting free MCPD permeated the Caco-2 monolayer most likely via a diffusion mechanism because their permeation profiles were independent of the dose. Similar permeation profiles were obtained for 2- and 3-MCPDs.
AIMS/HYPOTHESIS: Recently, incretin-related agents have been reported to attenuate insulin resistance in animal models, although the underlying mechanisms remain unclear. In this study, we investigated whether anagliptin, the dipeptidyl peptidase 4 (DPP-4) inhibitor, attenuates skeletal muscle insulin resistance through endothelial nitric oxide synthase (eNOS) activation in the endothelial cells. We used endothelium-specific Irs2-knockout (ETIrs2KO) mice, which show skeletal muscle insulin resistance resulting from a reduction of insulin-induced skeletal muscle capillary recruitment as a consequence of impaired eNOS activation. METHODS: In vivo, 8-week-old male ETIrs2KO mice were fed regular chow with or without 0.3% (wt/wt) DPP-4 inhibitor for 8 weeks to assess capillary recruitment and glucose uptake by the skeletal muscle. In vitro, human coronary arterial endothelial cells (HCAECs) were used to explore the effect of glucagon-like peptide 1 (GLP-1) on eNOS activity. RESULTS: Treatment with anagliptin ameliorated the impaired insulin-induced increase in capillary blood volume, interstitial insulin concentration and skeletal muscle glucose uptake in ETIrs2KO mice. This improvement in insulin-induced glucose uptake was almost completely abrogated by the GLP-1 receptor (GLP-1R) antagonist exendin-(9-39). Moreover, the increase in capillary blood volume with anagliptin treatment was also completely inhibited by the NOS inhibitor. GLP-1 augmented eNOS phosphorylation in HCAECs, with the effect completely disappearing after exposure to the protein kinase A (PKA) inhibitor H89. These data suggest that anagliptin treatment enhances insulin-induced capillary recruitment and interstitial insulin concentrations, resulting in improved skeletal muscle glucose uptake by directly acting on the endothelial cells via NO- and GLP-1-dependent mechanisms in vivo. CONCLUSIONS/INTERPRETATION: Anagliptin may be a promising agent to ameliorate skeletal muscle insulin resistance in obese patients with type 2 diabetes.
Streptomyces sp. strain 2238-SVT4 produces hatomarubigins A, B, C, and D, which belong to the angucycline family. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 30 open reading frames, including those for the minimal polyketide synthase, ketoreductase, aromatase, cyclase, O-methyltransferase, oxidoreductase, and oxygenase genes. Expression of a part of the gene cluster containing hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B, and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY, among the remaining genes.
Hepatic lipase (HL) plays a major role in the regulation of plasma lipids. Several groups seeking to find association between the gene encoding HL (LIPC) and plasma concentrations of high-density lipoprotein cholesterol (HDLc) using various methods and populations have reported conflicting results. We have approached the problem of demonstrating a relationship between the LIPC locus and HDLc by means of haplotype association using four single nucleotide polymorphisms (SNPs) (rs12594375G/A, rs8023503C/T, rs4775047C/T, and rs11634134T/A) located in intron 1 of the LIPC gene in two independent Japanese populations consisting of 2,970 and 1,638 individuals, respectively. Significant association between hyperalphalipoproteinemia and a specific haplotype in this intron was detected in both populations. When HDLc levels among the three haplotypic categories were analyzed [haplotype rs8023503C/rs12594375G (haplotype-1; H1) homozygotes (H1H1), haplotype rs8023503T/rs12594375A (haplotype-2; H2) homozygotes (H2H2), and heterozygotes (H1H2)], HDLc levels were lowest among H1H1 [mean +/- standard error (SE) = 58.4 +/- 0.4 mg/dl], highest among H2H2 (62.5 +/- 0.8 mg/dl), and intermediate among H1H2 (59.2 +/- 0.4 mg/dl) (P = 0.00011), indicating that H2 haplotype elevates plasma HDLc levels. This association was validated in the second population (n = 1,638) (P = 0.00070). The results provide convincing evidence that the LIPC locus influences HDL metabolism.
Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.
We examined the effects of pesticides on the central and peripheral nervous system in the setting of a tobacco farm at a developing country. Maximal motor and sensory nerve conduction velocities (MCV and SCV, respectively) in the median, sural and tibial nerves, postural sway, and brain-evoked potentials (auditory event-related and visual-evoked potentials) were measured in 80 male tobacco farmers and age- and sex-matched 40 controls in Kelantan, Malaysia. Median SCV (finger-wrist) in farmers using Delsen (mancozeb, dithiocarbamate fungicide), who showed significant decrease of serum cholinesterase activities, were significantly lower compared with the controls. Sural SCV in farmers using Fastac (alpha-cypermethrin, pyrethroid insecticide) and median MCV (elbow-wrist) in farmers using Tamex (butralin, dinitroaniline herbicide) were significantly slowed compared with their respective controls. In Delsen (mancozeb, dithiocarbamate) users, the power of postural sway of 0-1 Hz was significantly larger than that in the controls both in the anterior-posterior direction with eyes open and in the right-left direction with eyes closed. The former type of sway was also significantly increased in Tamaron (methamidophos, organophosphorus insecticide) users. In conclusion, nerve conduction velocities and postural sway seem to be sensitive indicators of the effects of pesticides on the central and peripheral nervous system.
        
Title: Acephate in biological fluids of two autopsy cases after ingestion of the chemical Tanaka T, Tanaka N, Kita T, Kasai K, Sato H Ref: J Forensic Science, 50:933, 2005 : PubMed
Two autopsy cases, where the individuals were suspected of having ingested acephate, an organophosphorous insecticide, are reported. Acephate and its active metabolite, methamidophos (MP), were analyzed in the biological fluids by GC/MS, using the salting out method with liquid-liquid extraction columns. The first case was that of a 70-year-old man whose blood acephate was 149 microg/mL, and MP was 3.0 microg/mL. Serum pseudocholinesterase (ChE) activity was inhibited. No remarkable finding of injury or disease was determined as the cause of his death, but acute poisoning by acephate was mostly suspected. The second case was that of a 60-year-old man. A deep gash in the left neck injured the left common carotid artery in addition to the severely ischemic state of the primary organs. His blood acephate was 46 microg/mL, and MP was not detected. ChE activity was in the normal range. Hemorrhage was mainly suspected as the cause of his death. The concentrations of acephate and MP in human blood after oral ingestion are first reported here, and the acute toxic level of acephate is discussed.
As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.
        
Title: Computerized posturography with sway frequency analysis: application in occupational and environmental health Yokoyama K, Araki S, Nishikitani M, Sato H Ref: Ind Health, 40:14, 2002 : PubMed
To examine the effects of occupational and environmental neurotoxicants on vestibular, cerebellar and spinocerebellar functions, the following three groups of subjects were examined, using a computerized posturography with sway frequency analysis: (1) 49 male chemical factory workers exposed to lead stearate, aged 27-63 (mean 43) years, with concurrent blood lead concentrations (BPbs) of 11-113 (mean 48) microg/100 g and past mean BPbs of 7-52 (mean 24) microg/100 g; (2) 29 male sandal, shoe and leather factory workers, aged 35-73 (mean 51) years, with urinary 2,5-hexanedione (HD) concentrations of 0.41-3.06 (mean 1.20) mg/g creatinine; and (3) 9 females, aged 19-58 (mean 29) years, who were exposed to sarin accidentally 6-8 months before the study (Tokyo Subway Sarin Poisoning, March 20,1995) and showed plasma cholinesterase (ChE) activities of 13-95 (mean 68) IU/l on the day of poisoning. The pattern of posturographic changes in lead workers suggested that the vestibulocerebellum (lower vermis), anterior cerebellar lobe and spinocerebellar afferent pathway were asymptomatically affected; the vestibulocerebellar change reflected concurrent lead absorption and the anterior cerebellar one reflected past absorption. Similarly, vestibulocerebellar and spinocerebellar functions were affected by n-hexane in solvent workers; the effect on the vestibulocerebellar function was probably inhibited by xylene. Also, the chronic (long-term) effect on the vestibulocerebellar function persisted in acute sarin poisoning. It is thus suggested that the vesitibulocerebellar function is most sensitive to all the three chemicals examined. It appears that the computerized posturography with frequency analysis is a useful technique for assessment of vestibular, cerebellar and spinocerebellar effects in occupational and environmental health.
The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features-an optimal C12-C8 chain length, pi-unsaturation introduction at the corresponding arachidonoyl Delta(8,9)/Delta(11,12) and oleoyl Delta(9,10) location, and an alpha-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with K(i)s that drop below 200 pM and are 10(2)-10(3) times more potent than the corresponding trifluoromethyl ketones.
We examined the influence of the concentration of sevoflurane and the degree of muscle block at the time of reversal on the activity of neostigmine. Ninety ASA 1-2 patients were anaesthetised with 0.2, 0.7 or 1.2 MAC of sevoflurane (30 patients each) in 66% nitrous oxide in oxygen. The electromyographic (EMG) response of the adductor digiti minimi was monitored at 20-s intervals after train-of-four stimulation of the ulnar nerve. The initial neuromuscular block was produced by vecuronium 100 micrograms.kg-1. When the amplitude of the first response (T1) values had recovered to 10%, 25% or 40% of the control, neostigmine 40 micrograms.kg-1 was administered. The train-of-four ratio values were recorded at 1-min intervals during the subsequent 15-min period. Higher endtidal concentrations (p < 0.0001) and more pronounced block at the time of reversal (p < 0.0001) were associated with a delayed recovery in the train-of-four ratio. In addition, the train-of-four ratio 15 min after neostigmine administration was more dependent on the sevoflurane concentration than on the degree of block present (p < 0.0001). These results confirm that neostigmine (40 micrograms.kg-1) can reverse vecuronium-induced but not sevoflurane-induced neuromuscular block.
1. To clarify possible regional and muscle layer differences in adrenergic innervation of swine myometrium, functional, biochemical and histochemical experiments were performed on longitudinal (LM) and circular (CM) muscle isolated from non-pregnant uteri of 84 gilts. 2. Transmural stimulation (TMS) in the presence of propranolol evoked tetrodotoxin-sensitive contractions in a frequency-dependent manner (2-20 Hz) in LM and CM. The cornual LM contractions were attenuated by phentolamine (1 microM) and by guanethidine (10 microM) though unaffected by atropine (1 microM). Contractions in cervical LM were diminished by atropine but not by phentolamine, and the corpus LM contractions were reduced incrementally by atropine and phentolamine when added sequentially. In CM, the TMS-induced contractions were abolished by tetrodotoxin and atropine in all three regions. 3. In response to noradrenaline (NA) and acetylcholine (ACh), LM contractile intensity was the most potent in cornua, slightly weaker in the corpus and weakest in the cervix. CM was insensitive to NA, and contractile responses elicited by ACh indicated no regional variation. 4. NA content, significantly greater in LM than in CM, was most highly concentrated in cornual LM. Nerves exhibiting glyoxylic acid-induced histofluorescence occurred in both LM and CM, though more abundantly in LM and with notable density in the cornual LM. Cholinesterase activity, distributed evenly throughout the three myometrial regions studied, was more intense in LM than in CM. 5. These results show that, in swine myometrium, innervation in cornual LM is predominantly noradrenergic, cervical LM is mostly cholinergic, and throughout the myometrium the CM layers are principally cholinergic.
        
Title: Failure to produce axon reflex-sweating in the volar skin of Japanese monkey (Macaca fuscata) and crab-eating monkey (Macaca irus) Aoki T, Narita T, Sato H, Izumi H Ref: Comparative Biochemistry & Physiology C, 79:325, 1984 : PubMed
The functional properties of the sweat glands and their innervation in the volar skin of three Japanese monkeys and two crab-eating monkeys were investigated. The sweat glands responded to both cholinomimetic and adrenomimetic agents, the former being highly predominant in the sudorific effect. Spontaneous emotional sweating was strongly or completely inhibited by atropine at 10(-8)-10(-7) g/ml, but not by dihydroergotamine at 10(-5)-10(-4) g/ml. Axon reflex sweating could not be produced by nicotine at 10(-5)-10(-4) g/ml in all of primates tested. The nerve fibers surrounding the sweat glands were histochemically confirmed to contain both acetyl- and butyrylcholinesterase.