5-Fluorouracil exacerbates cefepime-induced convulsions in pentylenetetrazol-kindled mice.

OBJECTIVE: The antibiotics cefepime and meropenem are recommended for the treatment of neutropenia. However, cefepime has been found to be associated with both peripheral and central adverse events such as renal impairment and seizures, respectively. Previous studies showed that cefepime exacerbated convulsions in corneal kindled mouse models of epilepsy. However, its involvement in chemotherapy-induced side effects is unknown. METHODS: In this study, we examined the convulsive potential of cefepime (500 mg/kg) and meropenem (500 mg/kg) in pentylenetetrazol (PTZ)-kindled mice using an electroconvulsive shock test with low-intensity stimulus currents. Then, the effects of 5-fluorouracil (5-FU, 200 and 400 mg/kg, i.p.) treatment, a model of chemotherapy-induced side effects, were investigated in the PTZ-kindled mouse model. RESULTS: In fully PTZ-kindled mice, intravenous administration of cefepime (500 mg/kg) or meropenem (500 mg/kg) did not elicit any convulsions in the electroconvulsive shock test with low-intensity stimulus currents. In the PTZ-kindled mice treated with 5-FU (200 mg/kg), intravenous administration of cefepime (500 mg/kg) exacerbated the convulsions that occurred within 1 min in the electroconvulsive shock test, and the mice subsequently developed convulsive status epilepticus. However, intravenous administration of meropenem (500 mg/kg) did not produce such effects. CONCLUSION: These findings suggest that the combination of 5-FU and cefepime exacerbates early-onset convulsive seizures and elicits delayed-onset convulsive status epilepticus. Additionally, 5-FU treatment increases the risk of induction of neurotoxic side effects by cefepime.

Change in mutation frequency at a TP53 hotspot during culture of ENU-mutagenised human lymphoblastoid cells.

Mutations in oncogenes or tumour suppressor genes cause increases in cell growth capacity. In some cases, fully malignant cancer cells develop after additional mutations occur in initially mutated cells. In such instances, the risk of cancer would increase in response to growth of these initially mutated cells. To ascertain whether such a situation might occur in cultured cells, three independent cultures of human lymphoblastoid GM00130 cells were treated with N-ethyl-N-nitrosourea to induce mutations, and the cells were maintained for 12 weeks. Mutant frequencies and spectra of the cells at the MspI and HaeIII restriction sites located at codons 247-250 of the TP53 gene were examined. Mutant frequencies at both sites in the gene exhibited a declining trend during cell culture and reached background levels after 12 weeks; this was also supported by mutation spectra findings. These results indicate that the mutations detected under our assay conditions are disadvantageous to cell growth.

TRPV1 mediates the anticonvulsant effects of acetaminophen in mice.

OBJECTIVE: Acetaminophen is one of the most commonly used analgesic and antipyretic drugs. It has been reported that acetaminophen has anticonvulsant effects in several animal models of seizure. An active metabolite of acetaminophen, AM404, inhibits the uptake of the endocannabinoid anandamide. However, the mechanism of the anticonvulsant effect of acetaminophen is unknown. METHODS: This study was performed to examine whether or not acetaminophen can protect against pentylenetetrazol-induced kindling in mice and to investigate the precise mechanisms of the anticonvulsant effect of acetaminophen using the fully kindled mouse models. RESULTS: Repeated administration of acetaminophen significantly delayed the progression of seizure severity induced by pentylenetetrazol. Additionally, acetaminophen showed a dose-dependent anticonvulsant activity against fully pentylenetetrazol-kindled seizures. AM404 also exhibited a dose-dependent anticonvulsant activity in fully kindled animals. The anticonvulsant activity of acetaminophen was antagonized by capsazepine and AMG9810, two transient receptor potential vanilloid-1 (TRPV1) antagonists. However, the transient receptor potential ankyrin 1 (TRPA1) antagonist HC030031 and CB1 receptor antagonist AM251 had no effect. CONCLUSION: These findings suggest that acetaminophen has an anticonvulsant effect in pentylenetetrazol-kindled mouse models and TRPV1 mediates the anticonvulsant action.

Environmental enrichment alleviates cognitive and behavioral impairments in EL mice.

Epilepsy in children is occasionally associated with comorbidities, such as cognitive impairment, behavioral disturbances, and social deficits. These neurobehavioral comorbidities are closely related to environmental factors and the severity of the seizures. Previous studies show that environmental enrichment has a beneficial effect in animal models of temporal lobe epilepsy following systemic chemoconvulsant administration. However, the effect of environmental enrichment on behavioral impairments in the EL mouse, a genetic model of human idiopathic epilepsy, remains unknown. In the present study, we examined the effect of environmental enrichment on cognitive and behavioral impairments in this murine model. The EL mice, under standard laboratory conditions, exhibited impairments in spatial memory in the Morris water maze test, hyperactivity and impaired habituation in the open-field test, and a deficit in social novelty preference in the three-chamber social approach test, compared with control DDY mice, a genetically related nonepileptic strain. These impairments in EL mice were ameliorated by exposure to an enriched environment. These findings suggest that environmental enrichment effectively ameliorates cognitive and behavioral deficits in EL mice.

Anticonvulsant effects of acetaminophen in mice: Comparison with the effects of nonsteroidal anti-inflammatory drugs.

OBJECTIVE: The appropriate use of analgesic drugs based on their degree of analgesia and adverse effects is important for pain management. Although it has been reported that AM404, a metabolite of acetaminophen, has anticonvulsant effects in several animal seizure models, little is known about the relation between acetaminophen and seizures. We therefore investigated the effects of acetaminophen on seizure susceptibility in several mouse seizure and epilepsy models and compared the effects with those of nonsteroidal anti-inflammatory drugs (NSAIDs). METHODS: Anticonvulsant activity was evaluated in ICR mice using maximum electroshock-induced seizure tests and acute pentylenetetrazol-induced seizure tests. Electrical kindling via corneal stimulation and pentylenetetrazol administration were used to establish animal kindling epilepsy models. Proconvulsive activity was examined using an electroconvulsive shock test with low-stimulus currents. RESULTS: Acetaminophen showed slight, but not statistically significant, anticonvulsant activity in both the maximum electroshock-induced seizure test (300-600mg/kg i.p.) and acute pentylenetetrazol-induced seizure test (100-600mg/kg i.p.). In contrast, acetaminophen exhibited significant anticonvulsant effects in corneal electroshock-kindled and pentylenetetrazol-kindled mice (ED50 values: 251 and 310mg/kg i.p., respectively). When the proconvulsive effects of NSAIDs were examined in the low-current electroconvulsive shock-induced seizure model, the nonselective cyclooxygenase (COX)-1 and COX-2 inhibitors indomethacin, diclofenac, and loxoprofen induced dose-dependent proconvulsant activity. Celecoxib, a COX-2 selective inhibitor, had no proconvulsant activity. CONCLUSION: These findings suggest that acetaminophen has a significant anticonvulsant effect and that its profile is completely different from that of NSAIDs.

trans-translation-mediated tight regulation of the expression of the alternative ribosome-rescue factor ArfA in Escherichia coli.

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

Mitochondrial HMG-CoA synthase partially contributes to antioxidant protection in the kidney of stroke-prone spontaneously hypertensive rats.

OBJECTIVE: Increased oxidative stress plays an important role in cardiovascular diseases including hypertension and stroke. Evidence has indicated that ketone bodies could exert antioxidative effects. We explored the role of renal mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS2) expression, a key control site of ketogenesis, in stroke-prone spontaneously hypertensive rats (SHRSPs) and their ancestral hypertensive but stroke-resistant spontaneously hypertensive rats (SHRs). METHODS: Two groups of SHRSPs were fed a standard chow or standard chow supplemented with clofibrate (an agonist of HMGCS2 promoter), respectively, and SHRs fed with a standard chow were used as controls. The renal levels of HMGCS2, Akt, and phosphorylated protein kinase B (Akt) were measured by western blotting. Malondialdehyde, catalase, superoxide dismutase, and glutathione peroxidase were detected by assay kits. RESULTS: Compared with SHRs, lower HMGCS2 protein expression, enhanced phosphorylated Akt signal, higher malondialdehyde levels, and higher catalase activity were observed in kidney tissues in SHRSPs (P < 0.05). No differences in superoxide dismutase and glutathione peroxidase activities were observed between SHRSPs and SHRs. Clofibrate treatment significantly upregulated renal HMGCS2 expressions, inhibited phosphorylation of Akt, and decreased malondialdehyde levels and catalase activities in SHRSP kidney tissues (P < 0.05). CONCLUSION: These results demonstrated the difference in HMGCS2 expression and oxidative stress in kidney tissues between SHRSPs and their SHR controls. The enhanced oxidative stress was partly due to the lower HMGCS2 expression regulated possibly by the Akt signaling pathway.

The role of cytochrome p-450 in salt-sensitive stroke in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) are salt sensitive: they develop severe hypertension and die of stroke within a short time after salt loading. We studied the role of cytochrome P-450 (CYP) isoforms in the brain and the effect of clofibrate to investigate the mechanism of salt sensitive stroke-proneness in SHRSP/Izm. Male SHRSP/Izm at 9 weeks of age were fed a regular diet with or without 0.25% clofibrate and given a 1% NaCl solution for drinking water for 10 d. The expression levels of CYP4A1, 2C11, and 2C23 were measured by Western blotting. Cerebral blood flow was measured with a laser Doppler method and blood vessel diameters were measured under microscopic observation. SHRSP/Izm died within 60 d after salt loading; however, clofibrate prolonged the survival (mean life span, 33+/-7 vs. 215+/-23 d, p<0.0001) without significant attenuation of the severe hypertension. CYP4A1 and CYP2C11 expression levels were lower in SHRSP/Izm than those in age-matched male spontaneously hypertensive rats (SHR/Izm) in the cerebral cortex (p<0.05). Salt loading down-regulated CYP2C11 expression in the cerebral cortex of SHRSP/Izm (p<0.05). No obvious change in cerebral CYP4A1 was observed in either salt-loaded SHRSP/Izm or SHR/Izm. Clofibrate significantly up-regulated the expression of cerebral CYP2C11 and significantly attenuated its salt-induced suppression (p<0.05). Additionally, clofibrate significantly increased blood vessel diameters (p<0.01) and cerebral blood flow (p<0.0001). CYP2C11 plays an important role in regulating cerebral blood flow and, as a result, in preventing stroke in the salt-sensitive stroke-prone SHRSP/Izm.

A novel aerobactin utilization cluster in Vibrio vulnificus with a gene involved in the transcription regulation of the iutA homologue.

We demonstrated that Vibrio vulnificus M2799 utilizes aerobactin for growth as an exogenous siderophore under iron-limiting conditions, concomitant with enhanced production of the 76-kDa iron-repressible outer membrane protein. Subsequently, by applying the Fur titration assay method to the M2799 genomic libraries followed by further cloning of the regions surrounding the candidate genes, we identified the 8.4-kb aerobactin utilization gene cluster which consists of five genes arranged in three distinct transcriptional units. It was confirmed by disruption of the corresponding genes that the first unit forming a three-gene operon (vatCDB) and the third unit of a single gene (iutA) encode an ATP-binding cassette transport component and the 76-kDa ferric aerobactin receptor, respectively. The second unit of another single gene (iutR), encodes a homologue of the GntR family of transcriptional repressors. Although transcription of the first and third units was iron-regulated, the iutR gene was transcribed regardless of iron status in the growth medium. Construction of an iutR disruptant coupled with genetic complementation experiments suggested that the gene encodes a transcriptional repressor for iutA. This is the first example of a regulator gene involved in aerobactin-enhanced production of IutA.

An iron-regulated gene required for utilization of aerobactin as an exogenous siderophore in Vibrio parahaemolyticus.

A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5.6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding (55)Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.

Induction of an outer membrane protein of 78 kDa in Vibrio vulnificus cultured in the presence of desferrioxamine B under iron-limiting conditions.

Although Vibrio vulnificus is known to be able to utilize ferrioxamine B as an iron source, its outer membrane receptor remains to be determined. In this study, we found that V. vulnificus expressed a new outer membrane protein of 78 kDa when grown in the presence of desferrioxamine B under iron-limiting conditions. The desferrioxamine B-dependent iron uptake was only observed in bacterial cells expressing this protein. Furthermore, non-denaturing polyacrylamide gel electrophoresis followed by autoradiography of the outer membrane preparation containing the 78-kDa protein preincubated with [(55)Fe]ferrioxamine B provided a single radioactive band in which the 78-kDa outer membrane protein was present as the major component. These lines of evidence suggest that the inducible 78-kDa protein may serve as the cell-surface receptor for ferrioxamine B.