Circadian rhythm of active amino acid transport in rat liver

Abstract

The circadian change of the encephalic photosensitivity of quail has been demonstrated. In this study the diurnal variation of the retinal photosensitivity was investigated by the electroretinogram (ERG) to explore the phase relation between the retinal and encephalic systems. The b‐wave, a major component of the ERG, was used as a measure of retinal photosensitivity.

1. In the bird maintained in LD12:12 the b‐wave amplitude of the ERG stayed at a high level for the first 10 h of the light period, and decreased abruptly around the time of light‐off. The decreased level continued until midnight. Thenceforth the b‐wave amplitude recovered progressively to the daytime level before the time of next light on.

2. In the bird maintained in LD16:8, the decrease of the retinal sensitivity in the light phase was initiated prior to the time of expected light‐off, and the onset time of decreasing tendency was advanced by 2 h, with dark adaptation for 30 min given immediately before the ERG measurement. Upon continuous light exposure, the b‐wave amplitude always remained at low level.

3. Periodic changes in retinal sensitivity persisted when the environmental dark phase was temporarily extended for 19 to 30 h.

These observations suggest that the diurnal rhythm of the neural retina in quail might be generated endogenously and appears to control the encephalic photosensitive system.     

生长猪结肠乳酸菌菌群的昼夜节律性变化

【目的】本文旨在研究生长猪在24 h内结肠乳酸菌的多样性变化、丰富度变化与节律性变化。【方法】选取6头装有结肠瘘管的“杜×长×大”三元杂交生长猪,于清晨6:00开始,每隔3 h连续采集一天内生长猪的结肠食糜,提取DNA,用乳酸菌特异性引物进行高通量测序,在属和种水平上分析结肠乳酸菌菌群丰度与节律性变化。【结果】生长猪结肠乳酸菌在24 h内Chao1和Simpson指数发生显著性变化(P<0.05);属水平上Lactobacillus相对丰度最高,一天内在6:00时相对丰度最低,为94.15%,18:00时相对丰度最高,为97.46%;种水平上Lactobacillus johnsonii相对丰度最高,一天内在3:00时相对丰度最低,为47.66%,18:00时相对丰度最高,为71.59%,Lactobacillus reuteri丰度次之。生长猪结肠乳酸菌中46个核心OTU具有节律性,均为Lactobacillus;而在种水平上Lactobacillus gasseri、Lactobacillus johnsonii、Lactobacillus sp. KC45a和Lactobacillus reuteri显现出节律性(P<0.05)。【结论】生长猪结肠乳酸菌在24 h内多样性发生显著性变化,在种水平上显现出节律性,丰富了我们对猪肠道微生物昼夜节律的了解。     

Polycomb homologs are involved in teratogenicity of valproic acid in mice

BACKGROUND: Valproic acid (VPA) is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its teratogenic mechanisms are unknown. We have attempted to describe a fundamental role of the Polycomb group (Pc-G) in VPA-induced transformations of the axial skeleton. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of vehicle or VPA (800 mg/kg) on gestation day (GD) 8. The expression of genes encoding Polycomb and trithorax groups was measured by quantitative real-time RT-PCR using total RNA isolated from the embryos exposed to vehicle or VPA for 1, 3, and 6 hr. In addition, the use of two less teratogenic antiepileptic chemicals valpromide (VPD) and valnoctamide (VCD) provide reliable evidence to support the relationship between VPA teratogenicity and the Polycomb group. RESULTS: At a teratogenic level, VPA inhibits the expression of the Polycomb group genes, including Eed, Ezh2, Zfp144, Bmi1, Cbx2, Rnf2, and YY1 in the mouse embryos. In contrast, neither VPD nor VCD have significant effects on the expression of those genes affected by VPA. The trithorax group (trx-G) gene MLL, which is known to be required to maintain homeobox gene expression such as the Polycomb gene, is not affected by a teratogenic dose of VPA. CONCLUSIONS: We propose that, during embryonic development, VPA may affect the gene silencing pathway mediated by the Polycomb group complex. The epigenetic mechanism of VPA teratogenicity on anteroposterior patterning is suspected.     

Amidic modification of valproic acid reduces skeletal teratogenicity in mice

BACKGROUND: The antiepileptic drug valproic acid (VPA) is well known to cause neural tube and skeletal defects in both humans and animals. The amidic VPA analogues valpromide (VPD) and valnoctamide (VCD) have much lower teratogenicity than VPA inducing exencephaly in mice. The objective of this study was to investigate the teratogenic effects of VPA, VPD, and VCD on the skeleton of NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of VPA (400 and 800 mg/kg), VPD (800 mg/kg), or VCD (800 mg/kg) on the morning of gestation day (GD) 8. Cesarean section was carried out on GD 18. Live fetuses were double‐stained for bone and cartilage and their skeletons were examined. RESULTS: Significant increases in fetal loss and exencephaly rate were observed with VPA at 800 mg/kg compared to the vehicle control. There were no significant differences between either VPD or VCD and the control groups for any parameter at cesarean section. A number of abnormalities were dose‐dependently induced at high incidences by VPA in both the cartilage and bone of vertebrae, ribs and sternum. In contrast, lower frequencies of abnormality were exhibited with VPD and VCD than VPA in all skeletons affected by VPA. CONCLUSIONS: These findings clearly indicate that VPD and VCD are distinctly less teratogenic than VPA in the induction of not only neural tube defects, but also skeletal abnormalities. A structure‐teratogenicity relationship of VPA on the skeleton is suspected. Birth Defects Res B 71:47–53, 2004. © 2004 Wiley‐Liss, Inc.     

Myo-inositol enhances teratogenicity of valproic acid in the mouse

BACKGROUND: Valproic acid (VPA) is an anticonvulsant drug that is widely used therapeutically for a variety of neurological conditions. VPA is also well known for its teratogenic potential in both humans and experimental animal models. The typical malformations observed following VPA exposure include neural tube defects (NTDs) and craniofacial and skeletal malformations. Nevertheless, the mechanisms underlying VPA's anticonvulsant efficacy or its teratogenicity remain to be elucidated. It was recently suggested that a relationship exists between VPA exposure and the cellular depletion of myo-inositol (INO). Furthermore, INO has been shown to rescue NTDs in the curly tail mouse. The aim of this study was to investigate the interactions of VPA and INO in the developing embryo. METHODS: For this purpose, 2 strains of mice were used: SWV/Fnn (known to be sensitive to VPA) and LM/Bc (known to be resistant to VPA-induced NTDs). Pregnant females were randomly assigned to 4 experimental groups: control, VPA (600 mg/kg), INO (400 mg/kg), and VPA plus INO. VPA was injected IP at 8.5 days postcoitum (dpc). INO was administered PO twice a day from 6.5 to 10.5 dpc. At term the dams were killed, the uteri were removed, and all of the general toxicological parameters (number of implants, resorptions, dam weight, and fetus weight) were recorded and statistically analyzed. RESULTS: Postimplantation loss in the SWV/Fnn strain and NTDs in the LM/Bc strain were significantly increased after the coadministration of VPA and INO. CONCLUSIONS: This work clearly indicates that INO enhances VPA-induced teratogenicity in the mouse.     

Intracellular distribution of hepatic copper in macular mutant mice

To evaluate the role of lysosomes in copper-mediated hepatocellular injury, copper was administered, sc, to both normal and macular mutant mice at doses of 4.5, 9.0, and 18 mg Cu/kg, and the subcellular distribution of copper has been investigated in the liver of normal and mutant mice 24h after injection. The amount of copper in all fractions of copper-treated mutant mice was markedly lower than those in copper-treated normal mice, with the exception of microsomal fraction. However, there were no distinct differences in the proportion of copper in subcellular fractions between normal and mutant mice.     

Lipidomic characteristics and clinical findings of epileptic patients treated with valproic acid

Our early study has found valproic acid (VPA)‐induced lipid dysmetabolism in animal model, however, the details of lipid profiling of VPA‐treated epileptic patients remain unknown. Therefore, in this study, the blood samples of VPA‐treated epileptic patients and VPA‐free controls were collected for lipidomic and biochemical assays. As results, clinical data showed the changes of some blood lipid molecules in VPA‐treated epileptic patients. In lipidomic assays, all 3797 annotated positive ions were identified prior to the data validation. In addition, the number of differentially expressed lipids were identified. And the 133 lipid molecules in VPA‐treated cases were significantly up‐regulated when compared to those in controls, while other 250 lipid metabolites were down‐regulated. Further, these lipid metabolites were mainly constituted with glycerolipids, glycerophopholipids, fatty acyls, sterol lipids. In addition, the most significant elevations of metabolite molecules of triglyceride, sphingomyelin, phosphorylcholine, ceramides, phenolic phthiocerol, as well as topped reductions of phosphoethanolamines, diradylglycerols, 1α,25‐dihydroxy‐24‐oxo‐22‐oxavitamin D3, 2‐deoxy‐20‐hydroxy‐5alpha‐ecdysone 3‐acetate, dolichyl‐4 phosphate were identified respectively. Taken together, these clinical findings demonstrate that negative impacts of exposure to VPA on expression of lipid mediators, progressively disrupting the functions of lipid molecules. Interestingly, these differentially expressed metabolites may be potential biomarkers for screening VPA‐induced dyslipidemia.     

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC₅₀) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (≥2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [¹⁴C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [¹⁴C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.     

Preliminary studies about novel strategies to reverse chemoresistance to adriamycin regarding glutathione metabolism,peroxisomal and extraperoxisomal hydroperoxide and valproic acid metabolic pathways

Summary— The present work was aimed at defining novel strategies to reverse chemoresistance to anticancer drugs, especially by interfering with cellular glutathione metabolism, peroxisomal and/or extraperoxisomal hydroperoxide metabolic pathways. Preliminary results are presented about molecules we demonstrated to be capable of interfering with hydrogen peroxide metabolism in cells. Prior to describing these molecules, a short overview of glutathione and free radical metabolic pathways is presented as well as a rapid presentation of the characteristics of chemo-sensitivity and -resistance towards the anticancer drug adriamycin, with special emphasis on hydrogen peroxide metabolism. The strategies currently developed to reverse chemoresistance are further presented. in subsequent sections, our own strategy to achieve inhibition of hydrogen peroxide breakdown and stimulation of peroxisomal hydrogen peroxide production is illustrated on the basis of molecular modelling studies and biochemical investigations on extraperoxisomal and peroxisomal metabolic pathways. Preliminary studies on cultured cells have been initiated. The perspective for future studies is presented as well as other possible models of chemoresistance as target for the design of hydrogen peroxide metabolism-interfering pharmacomolecules.     

丙戊酸与缺血性脑卒中相关研究进展

动脉粥样硬化和缺血性脑损伤是防治缺血性脑卒中所面临的两大难题,而细胞炎症损伤是它们的共同诱因。丙戊酸作为组蛋白去乙酰化酶抑制剂,具有抑制细胞炎症因子释放及保护神经的作用,所以丙戊酸可能是防治缺血性脑卒中的潜在治疗药物。本文从组蛋白去乙酰化酶对缺血性脑卒中的影响以及丙戊酸的抗炎机制两个方面进行综述。     

Circadian changes in endogenous concentrations of indole-3-acetic acid,melatonin, serotonin,abscisic acid and jasmonic acid in Characeae (Chara australis Brown)

Giant-celled Characeae (Chara australis Brown), grown for 4 months on 12/12 hr day/night cycle and summer/autumn temperatures, exhibited distinct concentration maxima in auxin (indole-3-acetic acid; IAA), melatonin and serotonin about 4 hr after subjective daybreak. These concentration peaks persisted after 3 day pretreatment in continuous darkness: confirming a circadian rhythm, rather than a response to “light on.” The plants pretreated for 3 d in continuous light exhibited several large IAA concentration maxima throughout the 24 hr. The melatonin and serotonin concentrations decreased and were less synchronized with IAA. Chara plants grown on 9/15 hr day/night cycle for 4 months and winter/spring temperatures contained much smaller concentrations of IAA, melatonin and serotonin. The IAA concentration maxima were observed in subjective dark phase. Serotonin concentration peaks were weakly correlated with those of IAA. Melatonin concentration was low and mostly independent of circadian cycle. The “dark” IAA concentration peaks persisted in plants treated for 3 d in the dark. The plants pretreated for 3 d in the light again developed more IAA concentration peaks. In this case the concentration maxima in melatonin and serotonin became more synchronous with those in IAA. The abscisic acid (ABA) and jasmonic acid (JA) concentrations were also measured in plants on winter regime. The ABA concentration did not exhibit circadian pattern, while JA concentration peaks were out of phase with those of IAA. The data are discussed in terms of crosstalk between metabolic pathways.     

Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice

BACKGROUND: Valproic acid (VPA) causes the failure of neural tube closure in newborn mice. However, the molecular mechanism of its teratogenesis is unknown. This study was conducted to investigate the genomewide effects of VPA disruption of normal neural tube development in mice. METHODS: Microarray analysis was performed on the head part of NMRI mouse embryos treated for 1 hr with VPA on gestational day (GD) 8. Subsequently, we attempted to isolate genes that changed in correlation with the teratogenic action of VPA by employing reduced teratogenic VPA analogs, valpromide (VPD) and valnoctamide (VCD), in a real-time PCR study. RESULTS: Microarray results demonstrated that during neurulation, many genes, some of whose functions are known and some unknown, were either increased or decreased after VPA injection. Some genes were affected by VPD or VCD in the same way as VPA, but others were not changed by the analogs. In this way, our system identified 11 increased and 20 decreased genes. Annotation analysis revealed that the increased genes included gadd45b, ier5, per1, phfl3, pou3f1, and sox4, and the decreased genes included ccne2, ccnl, gas5, egr2, sirt1, and zfp105. CONCLUSIONS: These findings demonstrate that expression changes in genes having roles in the cell cycle and apoptosis pathways of neural tube cells were strongly expected to relate to the teratogenic, but not antiepileptic, activity of VPA. Our approach has allowed the expansion of the catalog of molecules immediately affected by VPA in the developing neural tube.     

Effect of pH on domoic acid toxicity in mice

Domoic acid is a shellfish toxin which produces gastrointestinal distress, followed by neurological symptoms such as headache, confusion, disorientation and severe deficits in short-term memory. Domoic acid is an amino acid which contains three carboxylic groups, and one imino group, and its solubility, rate of absorption, and elimination would vary depending on the protonation of these groups at different pH's. We propose that domoic acid toxicity varies with pH of administered domoic acid solution. Domoic acid toxicity was measured in mice as the onset times for scratching behaviour, seizure activity, and death, after the intraperitoneal administration of domoic acid at different pH's. Results of the present study show that the scratching behaviour, seizure activity, and death, occurred at 12, 40, and 55 min, after intraperitoneal administration of domoic acid at pH 3.7. Apparently, the onset times for three types of behaviours were relatively long, and well separated from each ot her. Domoic acid toxicity was lowest at pH 3.7, and highest at pH 7.4, with intermediate toxicity at other pH's. The onset time of scratching behaviour was not influenced by pH of domoic acid solution at three different doses. In contrast, the onset times for seizure activity, and death were significantly affected by pH of domoic acid, toxicity being higher at pH 7.4 than at pH 3.7. The pH effect on domoic acid toxicity diminished as the dose of domoic acid was increased. In fact, at 14.5 mg/kg domoic acid toxicity was similar at both pH's of 3.7 and 7.4. It is concluded that in vivo toxicity of domoic acid varies depending on pH of the administered solution. The differential toxicity of domoic acid at different pH may be related to its solubility, rate of absorption, and elimination, depending on the degree of protonation of domoic acid molecule. Domoic acid toxicity would also vary depending on the age of animal, receptor sensitivity and density in different regions of brain. (Mol Ce ll Biochem 167: 179-185, 1997)     

Anticonvulsant profile and teratogenic evaluation of potent new analogues of a valproic acid urea derivative in NMRI mice

BACKGROUND: Valproic acid (VPA) is used to treat epilepsy and bipolar disorders, as well as for migraine prophylaxis. However, its clinical use is limited by two life-threatening side effects: hepatotoxicity and teratogenicity. To develop a more potent and safer second-generation VPA drug, the urea derivatives of four VPA analogs (2-ethyl-3-methylpentanoyl urea, 2-ethylhexanoyl urea, 2-ethyl-4-methylpentanoyl urea, and 2-methylbutanoyl urea) were synthesized. METHODS: Four CNS-active analogs of a VPA urea derivative testedthe anticonvulsant activity in the maximal electroshock seizure test (MES) and subcutaneous metrazol seizure threshold test (scMet). Teratogenic effects of these compounds were evaluated in NMRI mice susceptible to VPA-induced teratogenicity by comparison with VPA. RESULTS: All four VPA analogs showed superior anticonvulsant activity over VPA. Compared with VPA, which induced neural tube defects (NTDs) in fetuses at 1.8 and 3.6 mmol/kg, the analog derivatives induced no NTDs at any concentration up to 4.8 mmol/kg (except for a single abnormality at 3.6 mmol/kg with 2-ethyl-3-methylpentanoyl urea). Skeletal examination also revealed that the acylurea derivatives induced vertebral and rib abnormalities in fetuses markedly less frequently than VPA. Our results confirmed that the analogue derivatives are significantly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS-active VPA analogs containing a urea moiety, which have better anticonvulsant potency and lack teratogenicity, are good potential candidates as second-generation VPA antiepileptic drugs. Birth Defects Res (Part B) 86:394–401, 2009. © 2009 Wiley-Liss, Inc.     

Teratology study of derivatives of tetramethylcyclopropyl amide analogues of valproic acid in mice

BACKGROUND: Although valproic acid (VPA) is used extensively for treating various kinds of epilepsies, it is well known that it causes neural tube and skeletal defects in both humans and animals. The amide and urea derivatives of the tetramethylcylcopropyl VPA analogue, N-methoxy-2,2,3,3-tetramethylcyclopropanecarboxamide (N-methoxy-TMCD) and 2,2,3,3-tetramethylcyclopropanecarbonylurea (TMC-urea), were synthesized and shown to have a more potent anticonvulsant activity than VPA. The objective of this study was to investigate the teratogenic effects of these compounds in NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of either VPA, N-methoxy-TMCD, or TMC-urea at 1.8 and 3.6 mmol/kg on gestation day (GD) 8. Cesarean section was performed on GD 18. First, the live fetuses were examined to detect any external malformations, then their skeletons were double-stained for bone and cartilage and subsequently examined. RESULTS: Significant increases in fetal losses and neural tube defects were observed with administration of VPA at 3.6 mmol/kg when compared to the vehicle control. In contrast, upon cesarean section, there were no significant differences between either N-methoxy-TMCD or TMC-urea and the control groups for any parameter. Skeletal examination revealed that a number of the abnormalities were induced by VPA dose-dependently at high rates of incidence. These abnormalities were mainly at the axial skeletal level. However, lower frequencies of skeletal abnormality were observed with N-methoxy-TMCD and TMC-urea than with VPA. CONCLUSIONS: In addition to their more potent antiepileptic activity, these findings clearly indicate that N-methoxy-TMCD and TMC-urea are distinctly less teratogenic than VPA in NMRI mice.     

Circadian cortisol secretion and circadian adrenal responses to ACTH are maintained in dexamethasone suppressed capuchin monkeys (Cebus apella)

We tested the hypothesis that the capuchin monkey adrenal (Cebus apella) gland has oscillatory properties that are independent of adrenocorticotropic hormone (ACTH) by exploring under ACTH suppression by dexamethasone: (i) maintenance of a circadian rhythm of plasma cortisol and (ii) clock time dependency of plasma cortisol response to exogenous ACTH. The capuchin monkey had a clear ACTH and plasma cortisol rhythm. Dexamethasone treatment resulted in low non-rhythmic ACTH levels and decreased cortisol to 1/10 of control values; nevertheless, the circadian rhythm of plasma cortisol persisted. We found that cortisol response to exogenous ACTH was clock time-dependent. The maximal response to ACTH occurred at the acrophase of the cortisol rhythm (0800 h). These results suggest that the capuchin monkey adrenal cortex may possess intrinsic oscillatory properties that participate in the circadian rhythm of adrenal cortisol secretion and in the circadian cortisol response to ACTH.     

12-hour phase-shift of mice kidney rhodanese (thiosulfate sulfurtransferase) activity in the first two months of life

This study aimed to investigate and compare variation of renal rhodanese activity at 2^nd, 4^th and 8^th weeks of post-natal development (PND) in mice. The enzyme activity increased with age and was higher in females compared to males in all studied groups. Cosinor analysis revealed significant circadian rhythms (with period τ = 24 h) of enzyme activity in both genders with peak time shift during the PND. At the 2^nd week of PND (pre-weaning time), the circadian rhythm peaked at the beginning of light span, more precisely ≅1 HALO (Hours After Light Onset). A week after weaning (4^th week of PND), the peak time was located at the second half of photophase (≅9 HALO) in both genders. Four to six weeks later, about the 8^th week of PND, the circadian peak time was then recorded at ≅13 HALO. These findings suggest that rhodanese level and rhythm stabilization were age-dependent. Moreover, gender-related differences may stimulate discussions on the relationship between renal rhodanese and cyanide sensitivity.     

Circadian Rhythm of the Pupillary Response to Morphine and to Naloxone

Mice respond to morphine with characteristic mydriasis which is antagonized by naloxone. The present study presents data on the diurnal variation of these responses. The mydriatic response is at its highest level at 0800 and has a nadir at 2400. This effect is not influenced by ambient light conditions. The miotic response to naloxone in the morphine-dilated pupil is maximal between 1200 and 1600 and minimal at 0400. The curves of the responses to the two drugs, therefore, differ as do their acrophases. Pharmacokinetic factors are probably not responsible for the observed variations. It is speculated that these diurnal changes may reflect cyclic alteration in the affinity of the opiate receptors to agonists and antagonists, respectively, or in the number of available receptors.