Cycloheximide in the nanomolar range inhibits seed germination of Orobanche minor.

From the 992 samples of culture extracts of microorganisms isolated from soil in Japan, we found that the extract of Streptomyces sp. no. 226 inhibited Orobanche minor seed germination without significantly affecting the seed germination of Trifolium pratense and the growth of Aspergillus oryzae and Escherichia coli. Using ESI-MS, 1H-NMR, and 13C-NMR, we identified the active compound as cycloheximide. Cycloheximide had half-maximum inhibitory concentrations of 2.6 ng/mL for the inhibition of seed germination of O. minor and 2.5 µg/mL for that of the conidial germination of A. oryzae. Since cycloheximide is known to inhibit translation by interacting with ribosomal protein L28 (RPL28) in yeast, we investigated whether RPL protein of O. minor plays a critical role in the inhibition of O. minor seed germination. Our data suggested that O. minor RPL27A was not sensitive to cycloheximide by comparing it to the strain expressing S. cerevisiae RPL28. These findings suggest the presence of an unidentified mechanism by which cycloheximide hinders O. minor seed germination.

The role of increased FGF21 in VLDL-TAG secretion and thermogenic gene expression in mice under protein malnutrition.

Protein malnutrition promotes hepatic lipid accumulation in growing animals. In these animals, fibroblast growth factor 21 (FGF21) rapidly increases in the liver and circulation and plays a protective role in hepatic lipid accumulation. To investigate the mechanism by which FGF21 protects against liver lipid accumulation under protein malnutrition, we determined whether upregulated FGF21 promotes the thermogenesis or secretion of very-low-density lipoprotein (VLDL)-triacylglycerol (TAG). The results showed that protein malnutrition decreased VLDL-TAG secretion, but the upregulation of FGF21 did not oppose this effect. In addition, protein malnutrition increased expression of the thermogenic gene uncoupling protein 1 in inguinal white adipose and brown adipose tissue in an FGF21-dependent manner. However, surgically removing inguinal white adipose tissue did not affect liver triglyceride levels in protein-malnourished mice. These data suggest that FGF21 stimulates thermogenesis under protein malnutrition, but this is not the causative factor underlying the protective role of FGF21 against liver lipid accumulation.

Multi-residue determination of pesticides in green tea by gas chromatography-tandem mass spectrometry with atmospheric pressure chemical ionisation using nitrogen as the carrier gas.

Helium is commonly used as a carrier gas in gas chromatography-tandem mass spectrometry (GC-MS/MS); however, there are growing concerns regarding its global shortage and the resulting limited supply and high cost. Using nitrogen as an alternative carrier gas in GC-MS/MS with the widely used electron ionisation (EI) technique leads to a significantly lower sensitivity; thus, in this study, we explored the use of atmospheric-pressure chemical ionisation (APCI) as the ionisation method and examined the applicability of GC-(APCI)MS/MS with nitrogen gas for the determination of pesticide residues. GC-(APCI)MS/MS using nitrogen provided slightly wider peaks, and poorer isomeric separation compared to those using helium under identical conditions; however, the peak intensities were comparable. GC-(APCI)MS/MS using nitrogen was validated for 166 pesticides in green tea at a spiking level of 0.01 mg/kg and was compared with the conventional GC-(EI)MS/MS using helium gas. Except dimethomorph and resmethrin, GC-(APCI)MS/MS showed satisfactory results that were comparable to those of GC-(EI)MS/MS for most compounds, with trueness in the range of 73%-95% and relative standard deviations of <11%. The sensitivity and selectivity of GC-(APCI)MS/MS with nitrogen were superior to those of GC-(EI)MS/MS with helium. Therefore, GC-(APCI)MS/MS using nitrogen as the carrier gas, which has minimal concerns related to availability, could be a promising alternative to the conventional GC-(EI)MS/MS technique that employs helium.

Quantitative analysis of pesticide residues in tea by gas chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) using an atmospheric pressure chemical ionization (APCI) source was applied for the quantitative analysis of pesticide residues in tea. To determine the optimum ionization conditions for multiresidue analysis, the full-scan mass spectra and peak intensities of pesticides were compared in the presence and absence of water as a modifier. When water was added as a modifier in the ion source, most of the target compounds formed [M+H]+ ions and exhibited enhanced intensities. However, compounds consisting of only carbon, hydrogen, and chlorine, such as aldrin, γ-hexachlorocyclohexane, and p,p'-dichlorodiphenyldichloroethane, typically formed M+· or fragment ions, whose intensities were significantly decreased by the addition of water. GC-MS/MS methods using APCI (without modifier addition) and electron ionization (EI) were validated for 16 pesticides in tea at spiking levels of 0.01 and 0.1 mg/kg. Unlike EI, signal suppression was observed for most compounds at a spiking level of 0.01 mg/kg using APCI; however, dilution of the samples minimized this effect. Using APCI, the trueness of the target compounds ranged from 77% to 121% at both spiking levels, except for pyrethrins owing to matrix effects, with relative standard deviations of less than 14%. For most compounds, these results were comparable with those obtained using EI. However, because the use of APCI limited fragmentation, this ionization technique offered significantly higher sensitivity and specificity than EI. Using APCI, linear calibration curves with coefficients of determination greater than 0.998 were obtained in the range of 0.0005-0.5 µg/mL for all compounds. These findings indicated that GC-MS/MS with APCI is applicable for the routine monitoring of pesticide residues, even in complex samples such as tea.

A case of late onset riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency manifesting as recurrent rhabdomyolysis and acute renal failure.

We report an adult case of late-onset riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (MADD) characterized by episodic recurrent rhabdomyolysis and acute renal failure after the age of 46. Muscle biopsy revealed lipid storage myopathy and the finding of serum acylcarnitine and urine organic acid analyses were consistent with MADD. A compound heterozygous mutation was identified in the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene, including a novel missense mutation, which confirmed the diagnosis of MADD. After administration of riboflavin and L-carnitine, the muscle weakness and fatigability gradually improved. Acylcarnitine and urine organic acid were also normalized after supplementation. Thus, MADD should be included in one of the differential diagnoses for adult recurrent rhabdomyolysis. Gene analysis is useful to confirm the diagnosis, and early diagnosis is important because riboflavin treatment has been effective in a significant number of patients with MADD.

[Case of multiple sclerosis with "yes-yes" type head tremor].

A 32-year-old woman, who had developed head tremor and paresthesia of the right upper limb for several months, was admitted to our hospital The diagnosis of multiple sclerosis was made because the serial MRI showed multiple lesions in both the cerebral white matter and the cervical cord. Oligoclonal IgG band was positive. Her symptoms were improved by intravenous methylprednisolone and an antiepileptic drug (MEPM 1 g/day and CZP 1 mg/day). The head tremor was the so-called "yes-yes" type which shakes back and forth. Although this type of tremor has been considered to be developed by the lesions in the cerebellum, our patient seemed to develop the tremor by cervical cord lesion. Further investigation is needed to confirm the association of the head tremor and the cervical lesions in MS.

The glutamate release inhibitor riluzole attenuates the formation of conditioned place aversion induced by naloxone in rats undergoing a single morphine exposure.

Acute morphine exposure has been hypothesized to produce long-lasting central changes that contribute to the withdrawal aversion. We have most recently demonstrated that those changes may involve the glutamatergic system, including multiple classes of receptors. The present study was undertaken to further determine the involvement of the glutamatergic system by examining the effect of riluzole, a glutamate release inhibitor, on the motivational component of withdrawal from acute morphine dependence. The role of the amygdala in the action of riluzole was also assessed. We investigated the effects of riluzole on the conditioned place aversion (CPA) induced by naloxone-precipitated withdrawal from a single morphine exposure 24 h before, and on c-Fos expression within the amygdala during the withdrawal period in rats. Riluzole (2, 4, 8 mg/kg) dose-dependently attenuated the CPA without producing place conditioning itself. This result provided further evidence that glutamatergic mechanisms may be recruited in adaptational changes following acute morphine exposure and play a role in withdrawal aversion. In addition, riluzole appeared to produce nonspecific effects on c-Fos expression by itself, without specifically modifying c-Fos expression following naloxone-precipitated withdrawal in any region of the amygdala examined, suggesting that the amygdala may not serve as a specific site of action for riluzole.

Expression of c-Fos in the rat central amygdala accompanies the acquisition but not expression of conditioned place aversion induced by withdrawal from acute morphine dependence.

Conditioned reinforcement is hypothesized to be critically involved in drug addiction as a factor contributing to compulsive drug use and relapse. The present study focused on the neurobiology involved in the acquisition and expression of conditioned reinforcing effects of morphine withdrawal employing a conditioned place aversion (CPA) paradigm in acute-dependent rats. Expression of c-Fos in the amygdala (the central nucleus, CeA; the medial nucleus, MeA; the basolateral nucleus, BLA) following naloxone-precipitated withdrawal and the CPA test was examined using a range of naloxone doses (0.02, 0.05, 0.1, 0.2, 0.5 and 1.0 mg/kg). Naloxone dose-dependently produced CPA in rats given a single morphine exposure. In CeA, but not MeA with high-level constitutive neuronal activity, the naloxone-induced modification in c-Fos immunoreactivity following morphine pretreatment exhibited a dose-dependent pattern similar to that seen in the behavioral study. On the other hand, none of the three amygdaloid nuclei examined including CeA, MeA and BLA showed notable sensitivity of c-Fos to the conditioned withdrawal stimulus. These results suggest that CeA may play a role in the negative affective aspect of withdrawal from acute dependence, and in part suggest that the acquisition and expression of CPA may involve different neurobiological mechanisms.

Withdrawal-induced c-Fos expression in the rat centromedial amygdala 24 h following a single morphine exposure.

RATIONALE: An opiate antagonist was found to induce motivational withdrawal signs 24 h or even up to 48 h after a single dose of morphine in rats. OBJECTIVE: The present study was undertaken to determine whether such a withdrawal state would modify the neuronal activity in the brain. METHODS: A conditioned place aversion was established following a one-trial paradigm in rats undergoing a single exposure to morphine (10 mg/kg) 24 h prior to naloxone administration (0.5 mg/kg). Subsequently, the expression of the protein product of c-fos gene (c-Fos) following naloxone administration was measured within the extended amygdala. RESULTS: A significant increase in c-Fos immunoreactivity was seen in the centromedial amygdala (CMA), but not in the bed nucleus of the stria terminalis (BST) and the shell (AcbSh) of the nucleus accumbens (Acb) in rats treated with both morphine and naloxone. Further examination of the distribution of c-Fos-positive neurons along the rostrocaudal axis within CMA showed that the positive neurons distributed throughout this brain area and the caudal level of its central division (the central nucleus of the amygdala, CeA) exhibited the most robust labeling. CONCLUSIONS: Neuronal activity can be increased by naloxone at a dose that produces conditioned place aversion 24 h after a single morphine exposure. CMA, particularly the caudal level of its central division, was of high sensitivity. The current data also suggest a possible involvement of CMA in negative motivational component of precipitated withdrawal from acute morphine dependence.

Nicotine attenuates place aversion induced by naloxone in single-dose, morphine-treated rats.

RATIONALE: Acute physical dependence refers to the withdrawal syndrome precipitated by an opioid antagonist administered several hours after either a single dose or a short-term infusion of an opioid agonist. OBJECTIVES: We examined the mechanism of nicotine-induced attenuation of naloxone-precipitated withdrawal syndrome when used to produce an aversive motivational state in a place-conditioning paradigm. METHODS: The effect of nicotine was investigated through place aversion induced by naloxone in morphine-pretreated rats. Additionally, the mechanism of nicotine action in this model was explored specifically in relation to the dopaminergic system through the use of dopamine receptor antagonist and agonist. RESULTS: Place avoidance behavior was potently elicited by naloxone (0.5 mg/kg s.c.) 24 h after a single exposure to morphine (10 mg/kg s.c.). Avoidance behavior was attenuated by pretreatment with a 0.2-mg/kg dose of nicotine 15 min prior to naloxone administration. The effect of nicotine was completely blocked by mecamylamine, but not hexamethonium. The dopamine receptor antagonists haloperidol (0.05, 0.1 mg/kg, s.c.), SCH23390 (0.1 mg/kg, s.c.), raclopride (1.0 mg/kg, s.c.) and eticlopride (0.1 mg/kg, s.c.) showed effects similar to mecamylamine. Additionally, the dopamine receptor agonist apomorphine (0.03, 0.1, 0.3 mg/kg, s.c.) inhibited naloxone-induced place aversion in morphine-treated rats. CONCLUSION: The inhibitory effect of nicotine on place aversion induced by naloxone-precipitated morphine withdrawal may involve a dopaminergic portion of the central nervous system.