The prevalence of metabolic syndrome and its association with alanine aminotransferase in clozapine-treated Korean patients with schizophrenia.

Widely ranging prevalence rates for metabolic syndrome (MetS) in patients taking clozapine have been reported on the basis of various criteria, and most studies have been carried out in non-Asian countries. Therefore, we examined the prevalence of MetS in Korean patients using three commonly applied criteria with two waist-circumference cutoff values. The indirectly standardized prevalence ratio (ISPR) was estimated using data from the Fourth Korean National Health and Nutrition Examination Survey (KNHNES, 2007) to compare the prevalence of MetS in patients with that in the general population. In addition, we also examined whether serum alanine aminotransferase (ALT) and aspartate aminotransferase levels serve as biochemical markers for the identification of MetS. We reviewed the electromedical records of patients with schizophrenia who had taken clozapine as the sole antipsychotic for 3 months or more. The prevalence of MetS ranged from 34.5 to 46.9%, and the ISPR ranged from 2.4 to 2.8, given the three definitions of MetS and the two waist-circumference cutoff points for women. The ISPR for MetS among those aged 18-30 years was the highest and decreased with age in both men and women. After adjusting for age, patients with normal serum ALT levels who were in the top third were significantly more likely to have MetS compared with those who were in the bottom third. Logistic regression analysis showed that serum ALT levels and use of antidepressants were significantly related to the presence of MetS. Korean patients with schizophrenia who were receiving clozapine as the sole antipsychotic showed a high prevalence of MetS. Although we found substantial differences in the prevalence according to criteria, the ISPR indicated significantly higher rates of MetS in this group than in the general population. In the general population, younger patients had a much higher risk for MetS than older patients. Elevated levels of serum ALT that were in the normal range were associated with the presence of MetS, which suggests the possibility of using serum ALT level as an early indicator for MetS in patients treated with clozapine.

Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants.

AIM: This study has been aimed (i) to isolate and identify diazotrophs from Korean rice varieties; (ii) to examine the long-term effect of N and compost on the population dynamics of diazotrophs and (iii) to realize the shot-term inoculation effect of these diazotrophs on rice seedlings. METHODS AND RESULTS: Diazotrophic and heterotrophic bacterial numbers were enumerated by most probable number method and the isolates were identified based on morphological, physiological, biochemical and 16s rDNA sequence analysis. Long-term application of fertilizer N with compost enhanced both these numbers in rice plants and its environment. Bacteria were high in numbers when malate and azelaic acids were used as carbon source, but less when sucrose was used as a carbon substrate. The combined application promoted the association of diazotrophic bacteria like Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Gluconacetobacter diazotrophicus and Pseudomonas spp. in wetland rice plants. Detection of nifD genes from different diazotrophic isolates indicated their nitrogen fixing ability. Inoculation of a representative isolate from each group onto rice seedlings of the variety IR 36 grown in test tubes indicated the positive effect of these diazotrophs on the growth of rice seedlings though the percentage of N present in the plants did not differ much. CONCLUSIONS: Application of compost with fertilizer N promoted the diazotrophic and heterotrophic bacterial numbers and their association with wetland rice and its environment. Compost application in high N fertilized fields would avert the reduction of N(2)-fixing bacterial numbers and their association was beneficial to the growth of rice plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of high N fertilization on diazotrophic bacterial numbers could be reduced by the application of compost and this observation would encourage more usage of organic manure. This study has also thrown light on the wider geographic distribution of G. diazotrophicus with wetland rice in temperate region where sugarcane (from which this bacterium was first reported to be associating and thereon from other plant species) is not cultivated.

Electroconvulsive shock reduces inositol trisphosphate receptor1 mRNA in rat brain.

We studied the expression pattern of the inositol 1,4,5-trisphosphate receptor1 (InsP3R1) mRNA after a single electroconvulsive shock (ECS) in the rat brain by in situ hybridization. The expression was significantly decreased in the dentate gyrus and the CA1 area of the hippocampal formation 3 to 24 h after ECS. While the downregulation of InsP3R1 by accelerated protein degradation has been reported, our results indicate that the downregulation of InsP3R1 occurs at the mRNA level. This finding, along with our previous report on the InsP3 3-kinase(A), suggests that ECS regulates the phosphoinositide mediated signaling, which might be related to the therapeutic mechanism of ECS.

Isoform-specific changes of adenylate cyclase mRNA expression in rat brains following chronic electroconvulsive shock.

1. Electroconvulsive shock (ECS) has been reported to regulate the cAMP signaling system at various levels, suggesting that the cAMP system is involved in the therapeutic mechanism. 2. Chronic ECS has been suggested to change the expressions of adenylate cyclase (AC) genes, which constitute at least 9 families. However, little is known about its effect on the expression of AC. Therefore, to understand how chronic ECS alters the expression of AC genes in the brain, the authors analyzed the expression of 9 AC isoforms at the transcriptional level in rat hippocampus and cerebellum by quantitative RT-PCR following chronic ECS treatment. 3. Chronic ECS treatment was found to induce differential changes in the expression of AC isoforms in an isoform- and brain region-specific manner in the rat hippocampus and cerebellum. 4. Thus, it is concluded that chronic ECS induces differential changes in the expression of AC isoform mRNA in an isoform- and brain region-specific manner in the rat hippocampus and cerebellum. This suggests that the differential expression of AC isoforms might be an important mechanism by which chronic ECS treatment regulates the cAMP signaling system in rat brains.

Non-linear dynamic analysis of clozapine-induced electroencephalographic changes in schizophrenic patients--a preliminary study.

1. In order to find the electroencephalographic (EEG) parameters that reflect the effect of clozapine in schizophrenic patients, the authors applied various non-linear analyses on multi-channel EEG data drawn from patients before and after a therapeutic trial of clozapine. 2. The correlation dimension was difficult to extract from our limited time series EEG data and the authors did not find a meaningful association with clozapine use. The primary Lyapunov exponent could be reliably calculated but also did not reflect the effect of clozapine. 3. However, the mutual cross-prediction (MCP) algorithm showed potentially meaningful results. The driving system was shifted to the frontal channels after a 4-week trial with clozapine. Moreover, MCP might have a value as a predictor of treatment response. 4. Although preliminary in nature, the MCP might have greater power for interpreting complex changes from channel to channel in EEG induced by clozapine.

Electroconvulsive shock increases the phosphorylation of Pyk2 in the rat hippocampus.

Recently we reported the activation MAPKs, MEK, and Rafs by electroconvulsive shock (ECS) in the rat hippocampus. However, the upstream pathways for the activation of Raf-MEK-MAPK cascade after ECS have not been studied yet. Since the proline-rich tyrosine kinase 2 (Pyk2) and Src were reported to be involved in the activation of the MAPKs in neuronal cells, we examined tyrosine phosphorylation and activation of Pyk2 in the rat hippocampus after ECS. ECS transiently increased the phosphorylation of Pyk2 at multiple tyrosine residues (Tyr-402, Tyr-580, and Tyr-881). The phosphorylations reached the peak at 1 min and returned to basal level by 10 min after ECS. At 1 min after ECS, the binding of Pyk2 to Src and Grb2, and of Grb2 to Ras increased. These results suggested that ECS activates Pyk2, which then transmits the signal to MAPK cascade via Src, Grb2, and Ras in the rat hippocampus.

Induction of early growth response-1 gene expression by calmodulin antagonist trifluoperazine through the activation of Elk-1 in human fibrosarcoma HT1080 cells.

The early growth response gene-1 (Egr-1) is a transcription factor that plays an important role in cell growth and differentiation. It has been known that Egr-1 expression is down-regulated in many types of tumor tissues, including human fibrosarcoma HT1080 cells, and introduction of the Egr-1 gene into HT1080 cells inhibits cell growth and tumorigenic potential. Trifluoperazine (TFP), a phenothiazine class calmodulin antagonist, is known to inhibit DNA synthesis and cell proliferation and potentially important in antitumor activities. To understand the regulatory mechanism of Egr-1, we investigated the effect of TFP on expression of Egr-1 in HT1080 cells. Herein, we report that Egr-1 expression was increased by TFP in synergy with serum at the transcriptional level. Both the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN62 and the calcineurin inhibitor cyclosporin A enhanced TFP-dependent increase of Egr-1, suggesting that the Ca(2+)/calmodulindependent pathway plays a role in regulation of Egr-1 expression in HT1080 cells. The TFP-stimulated increase of the Egr-1 protein was preferentially inhibited by the MEK-specific inhibitor PD98059. In addition, activation of human Egr-1 promoter and the transcriptional activation of the ternary complex factor Elk-1 induced by TFP were inhibited both by pretreatment of PD98059 and by expression of the dominant-negative RasN17. These results indicate that the Ras/MEK/Erk/Elk-1 pathway is necessary for TFP-induced Egr-1 expression. We propose that the calmodulin antagonist TFP stimulates Egr-1 gene expression by modulating Ras/MEK/Erk and activation of the Elk-1 pathway in human fibrosarcoma HT1080 cells.

An N-methyl-D-aspartate antagonist, MK-801, preferentially reduces electroconvulsive shock-induced phosphorylation of p38 mitogen-activated protein kinase in the rat hippocampus.

Electroconvulsive shock (ECS) activates the mitogen-activated protein kinase (MAPK) family in the rat hippocampus, but the signaling pathways for this activation are not well understood. We investigated whether N-methyl-D-aspartate (NMDA) receptor mediated signaling is involved in the phosphorylation-activation of the MAPK family. The NMDA receptor antagonist, MK-801, dose-dependently reduced ECS-induced phosphorylation of p38 and its upstream kinase MKK6 up to 1 mg/kg. MK-801 also reduced the phosphorylation of ERK1/2 and MEK1, but only at high dosage, 2 mg/kg. Moreover, the reduction in the phosphorylation of p38 and MKK6 was greater than that of ERK1/2 and MEK1. Our results suggest that ECS activates p38 and ERK1/2 partly through an NMDA receptor-mediated signaling system in the rat hippocampus and that NMDA receptor mediated signaling is more responsible for the activation of the MKK6-p38 pathway than the MEK1-ERK pathway.

Differential activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinases by methyl methanesulfonate in the liver and brain of rats: implication for organ-specific carcinogenesis.

Methyl methanesulfonate (MMS), a direct-acting alkylating agent, is a strong brain carcinogen but a poor hepatocarcinogen in rats. To elucidate the mechanism(s) leading to tissue-specific carcinogenesis in response to MMS, we compared the activation of the stress-activated protein kinases (SAPKs), the c-Jun NH2-terminal kinase (JNK) and p38, in the liver and brain of rats after i.p. injection of MMS. p38 was activated in both the liver and brain, but JNK was activated only in the liver in a dose- and time-dependent manner. The activation of JNK was preceded by the activation of SAPK or extracellular signal-regulated protein kinase kinase 1/mitogen-activated protein kinase kinase 4 in the liver, but no activation of SAPK or extracellular signal-regulated protein kinase kinase 1/mitogen-activated protein kinase kinase 4 was observed in the brain. The activation of JNK in the liver was accompanied by increased phosphorylation of activating transcription factor 2 and followed by an increase in the phosphorylation and level of c-Jun protein, in contrast to no such changes in the brain. To study the physiological consequences of these differential molecular events in the liver and brain, we examined MMS-induced apoptosis, a process shown to involve stress kinase activation. A significant increase in apoptotic cell death was detected in the liver but not in the brain after a MMS injection, which correlated with the patterns of JNK activation in the liver. Taken together, our results demonstrate that a tissue-specific signaling pathway(s) leading to distinct physiological responses in the liver and brain of rats exposed to MMS exists, suggesting a possible explanation for tissue-specific carcinogenic effects exerted by MMS in vivo.

Electrocardiographic abnormalities in patients treated with clozapine.

BACKGROUND: Cardiovascular side effects of clozapine are not uncommon, but few systematic studies of these effects have been performed. In this study, we reviewed data on the electrocardiographic (ECG) abnormalities in patients treated with clozapine. METHOD: Sixty-one patients treated with clozapine were selected from the Seoul National University Hospital Treatment-Resistant Schizophrenia Clinic. A retrospective chart review was conducted to identify ECG abnormalities and cardiovascular side effects. RESULTS: The prevalence of ECG abnormalities in patients who had been using antipsychotics other than clozapine was 13.6% at baseline, which increased significantly to 31.1% after commencement of clozapine treatment. Among the 53 patients without baseline ECG abnormalities, 13 showed new-onset ECG abnormalities after using clozapine. Normal ECG under previous antipsychotic medication reduced the risk of new-onset ECG abnormalities, whereas increased age was found to increase the risk. The occurrence of orthostatic hypotension or tachycardia was not related to the development of ECG abnormalities. Most of the newly developed abnormalities had little clinical significance, and they tended to occur during the initial phase of treatment. In 10 patients, ECGs normalized despite the continued use of clozapine. Clozapine increased corrected QT interval (QTc) in a dose-dependent fashion; however, the clinical significance of this observation is uncertain. Pathologic prolongation of QTc was found to be rare. CONCLUSION: Although a substantial portion of patients treated with clozapine developed ECG abnormalities, most of the abnormalities were benign and did not hinder further treatment.

The activation of B-Raf and Raf-1 after electroconvulsive shock in the rat hippocampus.

We demonstrated that ECS activates the kinase activity of B-Raf and Raf-1 in the rat hippocampus. The activity was maximal at one minute after ECS and temporally coincided with the increased membrane translocation of Rafs and the reported activity of MAPK, but not with the phosphorylation of Rafs.

Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock.

Electroconvulsive shock (ECS), an effective treatment for psychiatric diseases, has been reported to induce immediate-early genes (IEGs) and to activate p42 and p44 MAPKs (ERK-1 and ERK-2) in rat brain. In this study, we examined the activation of the other members of MAPK family, c-Jun N-terminal protein kinase (JNK/SAPK) and p38. Following ECS, the phosphorylation of p38 was substantially increased in both hippocampus and cerebellum, but the increase of JNK phosphorylation was observed only in hippocampus. We also investigated the phosphorylation of their upstream kinases, SEK-1, MKK6 and MKK3. In both hippocampus and cerebellum, the phosphorylation of MKK6 showed closer correlation with p38 phosphorylation than that of MKK3. However, SEK-1, known as upstream kinase of JNK and p38 in vitro, corresponded with none of MAPKs. These results, with previous reports on the activation of ERK, indicate that ECS activates three MAPKs differentially in rat hippocampus and cerebellum, and suggest the possibility that unknown MAPKK may be involved in the activation of JNK in rat brain after ECS.

Inhibition of trifluoperazine-induced DNA fragmentation by cyclic AMP mediated signaling.

Trifluoperazine (TFP), a phenothiazine antipsychotic agent with calmodulin antagonist property, induces DNA fragmentation in a dose- and time-dependent manner in PC12 cells. Various agents affecting calcium mediated intracellular signal transduction such as calcium chelators, calcium ionopores, inhibitors of phospholipase C, and activators/inhibitors of protein kinase C did not block TFP-induced DNA fragmentation. Some of these agents themselves induced DNA fragmentation in the conditions under which they were examined. However, cholera toxin (selective Gs activator), forskolin (adenylate cyclase activator) or dibutyryl cyclic AMP (cyclic AMP analogue) inhibited TFP-induced DNA fragmentation in a dose-dependent manner. These results suggest that it is not the calcium but the Gs and adenylate cyclase pathways that play an important role in TFP-induced DNA fragmentation in PC12 cells.

Electroconvulsive shock does not induce c-fos and junB, but TIS1 and TIS8/zif-268, in neonatal rat hippocampus.

The induction in the animal brain of immediate early genes (IEGs) is known to be age-dependent, and it was suggested that, during neonatal period, signaling pathways for the induction of IEGs are immature. In this study, we investigated the induction of various IEGs in neonatal rat hippocampus after electroconvulsive shock (ECS). ECS did not induce c-fos and junB in the hippocampus of 7-day-old rat, but these genes were weakly induced at postnatal 14 days and to an adult level at postnatal 21 days; two other IEGs, TIS1 (NGFI-B, nur77) and TIS8 (zif-268, Egr-1, Krox-24, NGFI-A), were induced at postnatal 7 days, however. Our results suggested that during the neonatal period, signaling pathways for TIS1 and TIS8 induction in rat hippocampus after ECS are complete, while those for c-fos and junB are immature.

Electroconvulsive shock increases the phosphorylation of cyclic AMP response element binding protein at Ser-133 in rat hippocampus but not in cerebellum.

ECS increased the Ser-133 phosphorylation of CREB in rat hippocampus, but not in the cerebellum, even though the basal level of phosphorylated CREB was higher in cerebellum. These results indicate that c-fos induction after ECS may be mediated by Ser-133 phosphorylation of CREB in rat hippocampus, but not in the cerebellum.

Induction of tetradecanoyl phorbol acetate-inducible sequence (TIS) genes by electroconvulsive shock in rat brain.

We studied the induction of tetradecanoyl phorbol acetate-inducible sequences (TIS)1, 7, 8, 11, and 21 in rat cerebral cortex, hippocampus, and cerebellum after electroconvulsive shock (ECS). These genes were reported to be induced by depolarization in PC-12 cells. Single ECS induced TIS1, 8, 11, and 21, but not TIS7 genes in the rat brain regions examined. In cerebral cortex and hippocampus, induction of TIS1, TIS8, and TIS21 reached peak at 30 or 45 min after ECS. The induced mRNA of TIS1 and 21 decreased rapidly and returned almost to the basal level by 90 min after ECS, whereas those of TIS8 and 11 lasted longer. In cerebellum, TIS genes were induced and disappeared more rapidly than in the other two regions. The 10 and 20 daily ECSs did not affect the inducibility of TIS1, 11, and 21 in cerebellum, but the induction of TIS8 was attenuated by 35% after 20 daily ECSs. Our study indicated that ECS could induce some of the TIS genes in various rat brain regions, but the induction patterns were different depending on the TIS genes and brain regions. Our study also suggested that chronic ECS could not attenuate the induction of some immediate early genes.

Activation and tyrosine phosphorylation of 44-kDa mitogen-activated protein kinase (MAPK) induced by electroconvulsive shock in rat hippocampus.

Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42-kDa, but not 44-kDa, mitogen-activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42- and 44-kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phosphorylation on the tyrosine residue of 42-kDa MAPK in rat hippocampus after ECS, but not on that of 44-kDa MAPK. However, when we examined the immunoprecipitated 44-kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44-kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44-kDa MAPK, as well as 42-kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44-kDa MAPK than in 42-kDa MAPK.

Electroconvulsive shock reduces inositol 1,4,5-trisphosphate 3-kinase mRNA expression in rat dentate gyrus.

We investigated the expression of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA after a single electroconvulsive shock (ECS) with in situ hybridization histochemistry in rat brain. At 6 h after ECS, the expression was markedly decreased in the dentate gyrus, and the decrease was maintained until 9 h with a slight recovery. The InsP3 3-kinase mRNA content returned to basal levels after 12 h. We could not detect any apparent changes in the expression of InsP3 3-kinase mRNA in the CA1-CA3 areas of hippocampus, the striatum, and the cerebral cortex at any time point examined. In the temporal pattern, the reduction of the expression in the dentate gyrus was preceded by the induction of c-fos after ECS. These observations suggest that the InsP3 3-kinase might be one of the genes whose expression can be altered by ECS.

Symbiotic Potential, Competitiveness, and Serological Properties of Bradyrhizobium japonicum Indigenous to Korean Soils.

The symbiotic potential of Bradyrhizobium japonicum isolates indigenous to seven Korean soils was evaluated by inoculating soybeans with 10- and 1,000-fold-diluted soil suspensions (whole-soil inocula). At both levels, significant differences in the symbiotic potential of the indigenous B. japonicum isolates were demonstrated. The relationship between rhizobial numbers in the whole-soil inocula (x) and nitrogen fixation parameters (y) was best predicted by a straight line (y = a + bx) when the numbers in the inocula were 100 to 10,000 ml, while the power curve (y = ax) predicted the variation when the numbers were 1 to 100 ml. Thirty isolates from three soils showed wide differences in effectiveness (measured as milligrams of shoot N per plant), and several were of equal or greater effectiveness than reference strain B. japonicum USDA 110 on soybean cultivars Clark and Jangbaekkong. On both of the soybean cultivars grown in a Hawaiian mollisol, the Korean B. japonicum isolate YCK 213 and USDA 110 were of equal effectiveness; USDA 110 was the superior strain in colonization (nodule occupancy). Korean isolates YCK 117 and YCK 141 were superior colonizers compared with USDA 110. However, B. japonicum USDA 123 was the superior colonizer compared with isolates YCK 213, YCK 141, and YCK 117. In an immunoblot analysis of 97 indigenous Korean isolates of B. japonicum, 41% fell into the USDA 110 and USDA 123 serogroups. Serogroups USDA 110 and USDA 123 were represented in six of the seven soils examined. In one Korean soil, 100% of the B. japonicum isolates reacted only with antisera of YCK 117, an isolate from the same soil.

Oils as adhesives for seed inoculation and their influence on the survival of Rhizobium spp. and Bradyrhizobium spp. on inoculated seeds.

Mineral oil, peanut oil and soybean oil were compared with water and gum arabic for their suitability as adhesives for seed inoculation with peat inoculants. Inoculated seeds were stored at 4, 28 and 34°C, and sampled after 1, 3 and 9 days to determine the survival of rhizobia. Germination and nodulation tests were performed on the inoculated seeds. Results showed that oils were suitable adhesives for peat inoculants. Although the oils initially bound less inoculant to the seed, the number of surviving rhizobia was similar to that obtained by the gum arabic treatment after storage at 28 and 34°C for 3 and 9 days. An interesting finding of this experiment was that peanut and soybean oils were superior to gum arabic in supporting significantly higher numbers of chickpea rhizobia at 34°C. Inoculated seeds tested for germination and nodulation showed no adverse effects from the oil treatments. Oils hold good potential as adhesives for seed application in inoculation technology.