The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae.

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.

Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3' UTR regions.

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5' cap structure and the 3' poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3' untranslated region (3' UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3' UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3' UTR of CLB6 mRNA.

Pan2-Pan3 complex, together with Ccr4-Not complex, has a role in the cell growth on non-fermentable carbon sources.

There are two major deadenylase complexes, Ccr4-Not and Pan2-Pan3, which shorten the 3' poly(A) tail of mRNA and are conserved from yeast to human. We have previously shown that the Ccr4-mediated deadenylation plays the important role in gene expression regulation in the yeast stationary phase cell. In order to further understand the role of deadenylases in different growth condition, in this study we investigated the effect of deletion of both deadenylases on the cell in non-fermentable carbon containing media. We found that both ccr4Δ and ccr4Δ pan2Δ mutants showed similar growth defect in YPD media: when switched to media containing non-fermentable source (Glycerol-Lactate) only the ccr4Δ grew while the ccr4Δ pan2Δ did not. Ccr4, Pan2, and Pan3 were phosphorylated in GlyLac medium, suggesting that the activities of Ccr4, Pan2, and Pan3 may be regulated by phosphorylation in response to change of carbon sources. To get insights how Ccr4 and Pan2 function in the cell growth in media containing non-fermentable source only, we isolated multicopy suppressors for the growth defect on YPGlyLac media of the ccr4Δ pan2Δ mutant and identified two genes, STM1 and REX2, which encode a ribosome-associated protein and a 3'-5' RNA exonuclease, respectively. Our results suggest that the Pan2-Pan3 complex, together with the Ccr4-Not complex, has important roles in the growth on non-fermentable carbon sources.

Pbp1, the yeast ortholog of human Ataxin-2, functions in the cell growth on non-fermentable carbon sources.

Pbp1, the yeast ortholog of human Ataxin-2, was originally isolated as a poly(A) binding protein (Pab1)-binding protein. Pbp1 regulates the Pan2-Pan3 deadenylase complex, thereby modulating the mRNA stability and translation efficiency. However, the physiological significance of Pbp1 remains unclear since a yeast strain harboring PBP1 deletion grows similarly to wild-type strain on normal glucose-containing medium. In this study, we found that Pbp1 has a role in cell growth on the medium containing non-fermentable carbon sources. While the pbp1Δ mutant showed a similar growth compared to the wild-type cell on a normal glucose-containing medium, the pbp1Δ mutant showed a slower growth on the medium containing glycerol and lactate. Microarray analyses revealed that expressions of the genes involved in gluconeogenesis, such as PCK1 and FBP1, and of the genes involved in mitochondrial function, such as COX10 and COX11, were decreased in the pbp1Δ mutant. Pbp1 regulated the expressions of PCK1 and FBP1 via their promoters, while the expressions of COX10 and COX11 were regulated by Pbp1, not through their promoters. The decreased expressions of COX10 and COX11 in the pbp1Δ mutant were recovered by the loss of Dcp1 decapping enzyme or Xrn1 5'-3'exonuclease. Our results suggest that Pbp1 regulates the expressions of the genes involved in gluconeogenesis and mitochondrial function through multiple mechanisms.

Pbp1 mediates the aberrant expression of genes involved in growth defect of ccr4∆ and pop2∆ mutants in yeast Saccharomyces cerevisiae.

CCR4 and POP2 genes encode the catalytic subunit of the Ccr4-Not complex involved in shortening mRNA poly(A) tail in Saccharomyces cerevisiae. The ccr4Δ and pop2∆ mutants exhibit pleiotropic phenotypes such as slow and temperature-sensitive growth, aberrant expression of glucose repression genes and abnormal cell wall synthesis. We previously found that the growth defect of the ccr4Δ and pop2∆ mutants is suppressed by deletion of the PBP1 gene, which encodes poly(A)-binding protein (Pab1)-binding protein 1. In this study, we investigated the functional relationship between Ccr4/Pop2 and Pbp1 by measuring changes in gene expression in ccr4Δ and pop2∆ single mutants and ccr4Δ pbp1∆ and pop2∆ pbp1∆ double mutants. We found that expression of HSP12, HSP26, PIR3, FUS1 and GPH1 was increased in ccr4Δ and pop2∆ single mutants. The pbp1∆ mutation not only restored the growth defect but also reduced the increased expression of those genes found in the ccr4Δ and pop2∆ mutants. Over-expression of PBP1 in the ccr4Δ mutant further increased the expression of HSP12, HSP26, PIR3 and FUS1 and exacerbated the cell growth. These results suggest that the aberrant expression of a subset of genes, which is facilitated by Pbp1, contributes to the pleiotropic phenotypes of the ccr4Δ and pop2∆ mutants.

The eIF4E-binding protein Eap1 has similar but independent roles in cell growth and gene expression with the cytoplasmic deadenylase Ccr4.

eIF4E-binding proteins (4E-BPs) are translational repressors that compete with eIF4G for binding to eIF4E. Here we investigated the roles of yeast 4E-BPs, Eap1, and Caf20 in cell wall integrity pathway and gene expression. We found that eap1∆ mutation, but not caf20∆ mutation, showed synthetic growth defect with mutation in ROM2 gene encoding Rho1 GEF. The eap1∆ mutation also showed synthetic lethality with mutation in CCR4 gene encoding cytoplasmic deadenylase. Ccr4 functions in the degradation of LRG1 mRNA encoding Rho1 GAP. Eap1-Y109A L114A, which could not bind to eIF4E, did not suppress the synthetic lethality of eap1∆ ccr4∆ mutant, suggesting that 4E-binding of Eap1 is important for its function. We also found that eap1∆ mutant showed the derepression of stress response gene HSP12. 4E-binding of Eap1 was also required for the repression of HSP12 expression. Our results indicate that Eap1 has similar but independent roles in cell growth and gene expression with Ccr4.

Regulation of LRG1 expression by RNA-binding protein Puf5 in the budding yeast cell wall integrity pathway.

The PUF RNA-binding protein Puf5 is involved in regulation of the cell wall integrity (CWI) pathway in yeast. Puf5 negatively regulates expression of LRG1 mRNA, encoding for a GTPase-activating protein for Rho1 small GTPase. Here, we further analyzed the effect of Puf5 on LRG1 expression, together with Ccr4 and Pop2 deadenylases, Dhh1 decapping activator, and other PUF proteins. We found that the growth defect of puf5∆ mutant was enhanced by ccr4∆ mutation, which was partially suppressed by LRG1 deletion. Consistently, Lrg1 protein level was much more up-regulated in ccr4Δ puf5Δ double mutant than in each single mutant. Interestingly, LRG1 poly(A) tail length was longer in ccr4∆ mutant but not in puf5∆ mutant. Thus, Puf5 regulates LRG1 expression independently from Ccr4, although Puf5 recruits the Ccr4-Not deadenylase complex for mRNA destabilization. Unexpectedly, puf6Δ mutation suppressed the growth defect caused by ccr4Δ puf5∆ mutation. Loss of Rpl43a and Rpl43b ribosomal proteins, the previously identified Puf6 interactors, also suppressed the growth defect of ccr4Δ puf5Δ mutant. Our results suggest that Puf5 functions in the CWI pathway by regulating LRG1 expression in a deadenylase-independent manner, and that Puf6 is involved in the Ccr4- and Puf5-mediated regulation of cell growth through association with Rpl43.

Different Regulations of ROM2 and LRG1 Expression by Ccr4, Pop2, and Dhh1 in the Saccharomyces cerevisiae Cell Wall Integrity Pathway.

Ccr4, a component of the Ccr4-Not cytoplasmic deadenylase complex, is known to be required for the cell wall integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae. However, it is not fully understood how Ccr4 and other components of the Ccr4-Not complex regulate the CWI pathway. Previously, we showed that Ccr4 functions in the CWI pathway together with Khd1 RNA binding protein. Ccr4 and Khd1 modulate a signal from Rho1 small GTPase in the CWI pathway by regulating the expression of ROM2 mRNA and LRG1 mRNA, encoding a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP) for Rho1, respectively. Here we examined the possible involvement of the POP2 gene encoding a subunit of the Ccr4-Not complex and the DHH1 gene encoding a DEAD box RNA helicase that associates with the Ccr4-Not complex in the regulation of ROM2 and LRG1 expression. Neither ROM2 mRNA level nor Rom2 function was impaired by pop2Δ or dhh1Δ mutation. The LRG1 mRNA level was increased in pop2Δ and dhh1Δ mutants, as well as the ccr4Δ mutant, and the growth defects caused by pop2Δ and dhh1Δ mutations were suppressed by lrg1Δ mutation. Our results suggest that LRG1 expression is regulated by Ccr4 together with Pop2 and Dhh1 and that ROM2 expression is regulated by Khd1 and Ccr4, but not by Pop2 and Dhh1. Thus, Rho1 activity in the CWI pathway is precisely controlled by modulation of the mRNA levels for Rho1-GEF Rom2 and Rho1-GAP Lrg1. IMPORTANCE We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex.

Analysis of the Physiological Activities of Scd6 through Its Interaction with Hmt1.

Scd6, a yeast homologue of human RAP55, is a component of messenger ribonucleoproteins (mRNPs) that repress translation by binding to translation initiation factors, and also is a decapping activator along with the binding partners Edc3 and Dhh1. Herein, we report that Scd6 is a substrate of the intrinsic protein arginine methyltransferase, Hmt1, in budding yeast Saccharomyces cerevisiae. Mass spectrometric analysis revealed that several arginine residues within the Scd6 RGG motif, which is important for mRNA binding, were methylated in Hmt1 dependent manner. Under stress conditions such as glucose starvation, Scd6 localized to cytoplasmic processing bodies (P-bodies) wherein translationally repressed mRNPs and untranslated mRNAs accumulate. Localization of Scd6 to P-bodies was impaired in hmt1 deletion mutant and in the presence of methylation-deficient substitution of Scd6. In addition, deletion of scd6 and dhh1 led to severe synthetic growth defect at high temperature. Methylation-deficient mutation of Scd6 suppressed the phenotypic defects of scd6 dhh1 double mutant, whereas methylation-mimic mutation did not, suggesting that the arginine methylation might negatively regulate Scd6 function relating to Dhh1. Therefore, the present data suggest that Hmt1-based arginine methylation is required for Scd6 localization and function.

Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides.

We clarified the involvement of constitutive androstane receptor (CAR) in triazole-induced liver hypertrophy and tumorigenesis using CAR-knockout (CARKO) mice. Seven-week-old male CARKO and wild-type (WT) mice were treated with 200 ppm cyproconazole (Cypro), 1500 ppm tebuconazole (Teb), or 200 ppm fluconazole (Flu) in the diet for 27 weeks after initiation by diethylnitrosamine (DEN). At weeks 4 (without DEN) and 13 (with DEN), WT mice in all treatment groups and CARKO mice in Teb group revealed liver hypertrophy with mainly Cyp2b10 and following Cyp3a11 inductions in the liver. Teb also induced Cyp4a10 in both genotypes. Cypro induced slight and duration-dependent liver hypertrophy in CARKO mice. At week 27, Cypro and Teb significantly increased eosinophilic altered foci and/or adenomas in WT mice. These proliferating lesions were clearly reduced in CARKO mice administered both compounds. The eosinophilic adenomas caused by Flu decreased in CARKO mice. The present study indicates that CAR is the main mediator of liver hypertrophy induced by Cypro and Flu, but not Teb. In contrast, CAR played a crucial role in liver tumor development induced by all three triazoles.

Inhibitory potential of postnatal treatment with cyclopamine, a hedgehog signaling inhibitor, on medulloblastoma development in Ptch1 heterozygous mice.

Medulloblastomas (MBs) are thought to be derived from granular cell precursors in the external granular layer (EGL) of the developing cerebellum. Heterozygous patched1 (Ptch1) knockout mice develop MBs that resemble those in humans when the sonic hedgehog (Shh) signaling pathway is activated. The present study was conducted to evaluate postnatal effects of a Shh signaling inhibitor, cyclopamine, on the development of MBs in Ptch1 mice. Ptch1 and wild-type mice were treated daily with subcutaneous cyclopamine at 40 mg/kg or vehicle from postnatal day (PND) 1 to PND14, and the subsequent development of MBs and preneoplastic lesions was examined up to week 12 (W12). Proliferative lesions in the cerebellum, MBs, and preneoplastic lesions were only detected in Ptch1 mice. Cyclopamine treatment resulted in a statistically significant reduction in the incidence and/or area of proliferative lesions at PND14 and 21. The trend of decreasing preneoplastic lesions persisted up to W12. At PND7, cyclopamine treatment reduced the width and proliferation of the EGL regardless of genotype. These results indicate that inhibition of Shh signaling during cerebellar development has prolonged inhibitory potential on MB development in Ptch1 mice. This inhibitory potential might be related to inhibition of EGL proliferation, including preneoplastic MB cells.

Effects of pyperonyl butoxide on the female reproductive tract in rats.

This study was investigated the effects of piperonyl butoxide (PBO) on the female reproductive tract. Female Crj:Donryu rats were fed a basal diet containing 5,000, 10,000 or 20,000 ppm PBO for 28 days, and compared with food-restricted rats of comparable body weights to those in the PBO 10,000 or 20,000 ppm groups. Although treatment with 20,000 ppm PBO for 28 days depressed body weight gain, the abnormal estrous cyclicity, mainly prolonged diestrus, was also induced by the PBO treatment which was not correlated with body weight change. 20,000 ppm PBO treatment markedly decreased uterine weights and slightly decreased ovarian weights. 10,000 and 20,000 ppm PBO treatment increased liver weights. These cycle and organ weight changes were linked to atrophic uterus and increased atretic follicles in the ovary. In hormone assays, PBO at both doses reduced serum E2 levels, but did not affect corticosterone levels. An anti-uterotrophic assay showed a slight but significant decrease in absolute uterine weight and a reduction of endometrial epithelium height in the 20,000 ppm group. PBO was positive in an ER α antagonist reporter gene assay, although the activity was much weaker than that of 4-hydroxytamoxifen. These results indicate that high-dose PBO treatment directly induces atrophic changes in the female reproductive tract in rats, and these effects are likely the result of a hypoestrogenic state and the anti-estrogenic activity of PBO.

Dose-response involvement of constitutive androstane receptor in mouse liver hypertrophy induced by triazole fungicides.

To clarify the dose-response relationship between constitutive androstane receptor (CAR) activity and induction of cytochrome P450 2B (CYP2B) expression and hypertrophy by triazole fungicides in mouse liver, three dose levels of cyproconazole (Cypro), tebuconazole (Teb), fluconazole (Flu), and phenobarbital (PB), a typical CYP2B inducer, were administrated in diet to male wild-type (WT) and CAR-knockout (CARKO) mice for one week. In WT mice, all compounds dose-dependently induced liver weight increases and hepatocellular hypertrophy accompanied by CYP2B expression. In CARKO mice, these effects were not induced by PB, while Cypro or Flu induced these effects only at the highest dose. Dose-dependent liver hypertrophy was detected in CARKO mice treated with Teb, but at the lowest dose the intensity was weakened compared to WT mice. The present results indicate that Cypro and Flu mainly induced CAR-mediated liver hypertrophy, while Teb slightly involved CAR. The involvement of CAR in triazole-induced liver hypertrophy was dose-responsive. In addition, all three triazoles have non-CAR-mediated liver hypertrophy pathways, indicating that the hypertrophy induced by these triazoles differs from that of PB.

Pbp1 is involved in Ccr4- and Khd1-mediated regulation of cell growth through association with ribosomal proteins Rpl12a and Rpl12b.

The Saccharomyces cerevisiae Pbp1 [poly(A)-binding protein (Pab1)-binding protein] is believed to be involved in RNA metabolism and regulation of translation, since Pbp1 regulates a length of poly(A) tail and is involved in stress granule (SG) formation. However, a physiological function of Pbp1 remains unclear, since the pbp1Δ mutation has no obvious effect on cell growth. In this study, we showed that PBP1 genetically interacts with CCR4 and KHD1, which encode a cytoplasmic deadenylase and an RNA-binding protein, respectively. Ccr4 and Khd1 modulate a signal from Rho1 in the cell wall integrity pathway by regulating the expression of RhoGEF and RhoGAP, and the double deletion of CCR4 and KHD1 confers a severe growth defect displaying cell lysis. We found that the pbp1Δ mutation suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. The pbp1Δ mutation also suppressed the growth defect caused by double deletion of POP2, encoding another cytoplasmic deadenylase, and KHD1. Deletion of the gene encoding previously known Pbp1-interacting factor Lsm12, Pbp4, or Mkt1 did not suppress the growth defect of the ccr4Δ khd1Δ mutant, suggesting that Pbp1 acts independently of these factors in this process. We then screened novel Pbp1-interacting factors and found that Pbp1 interacts with ribosomal proteins Rpl12a and Rpl12b. Similarly to the pbp1Δ mutation, the rpl12aΔ and rpl12bΔ mutations also suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. Our results suggest that Pbp1 is involved in the Ccr4- and Khd1-mediated regulation of cell growth through the association with Rpl12a and Rpl12b.

Thickened area of external granular layer and Ki-67 positive focus are early events of medulloblastoma in Ptch1⁺/⁻ mice.

Patched1 (Ptch1) encodes a receptor for Sonic hedgehog (Shh) and is major gene related to human medulloblastoma (MB) in the Shh subgroup. MB is thought to arise from residual granule cell precursors (GCPs) located in the external granular layer (EGL) of the developing cerebellum. As the detailed preneoplastic changes of MB remain obscure, we immunohistochemically clarified the derived cell, early events of MBs, and the cerebellar developmental processes of Ptch1(+/-) (Ptch1) mice, an animal model of human MB of the Shh subgroup. In Ptch1 mice, the earliest proliferative lesions were detected at PND10 as focal thickened areas of outer layer of the EGL. This area was composed of GCP-like cells with atypia and nuclei disarrangement. In the latter cerebellar developmental period, GCP-like cell foci were detected at high incidence in the outermost area of the cerebellum. Their localization and morphological similarities indicated that the foci were derived from GCPs in the EGL. There were two types of the foci. A Ki-67-positive focus was found in Ptch1 mice only. This type resembled the GCPs in the outer layer of EGL characterized by having proliferating activity and a lack of neuronal differentiation. Another type of focus, Ki-67-negative, was observed in both genotypes and exhibited many of the same features of mature internal granule cells, suggesting that the focus had no preneoplastic potential. Due to morphological, immunohistochemical characteristics, our results indicate that the focal thickened area of EGL and Ki-67-positive foci are preneoplastic lesions of MB.

Development of an early induction model of medulloblastoma in Ptch1 heterozygous mice initiated with N-ethyl-N-nitrosourea.

Mice heterozygous for the ptch1 gene (ptch1 mice) are known as a valuable model of medulloblastoma, a common brain tumor in children. To increase the incidence and reduce the time required for tumor development, allowing for evaluation of modifier effects on medulloblastoma in a short time, we attempted to develop an early induction model of medulloblastoma in ptch1 mice initiated with N-ethyl-N-nitrosourea (ENU). Ptch1 mice and their wild-type littermates received a single intraperitoneal injection of ENU (10, 50 or 100 mg/kg) on postnatal day 1 (d1) or 4 (d4), and histopathological assessment of brains was conducted at 12 weeks of age. The width of the external granular layer (EGL), a possible origin of medulloblastoma, after injection of 100 mg ENU on d1 or d4 was measured in up to 21-day-old mice. Cerebellar size was apparently reduced at the 50 mg dose and higher regardless of genotype. Microscopically, early lesions of medulloblastomas occurred with a high incidence only in ptch1 mice receiving 10 mg on d1 or d4, but a significant increase was not observed in other groups. Persistent EGL cells and misalignment of Purkinje cells were increased dose-dependently. Although EGL was strikingly decreased after ENU injection, strong recovery was observed in mice of the d1-treated group. In summary, neonatal treatment with ENU is available for the induction of medulloblastoma in ptch1 mice, and 10 mg of ENU administered on d1 appeared to be an appropriate dose to induce medulloblastoma.

RNA-binding protein Khd1 and Ccr4 deadenylase play overlapping roles in the cell wall integrity pathway in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae RNA-binding protein Khd1/Hek2 associates with hundreds of potential mRNA targets preferentially, including the mRNAs encoding proteins localized to the cell wall and plasma membrane. We have previously revealed that Khd1 positively regulates expression of MTL1 mRNA encoding a membrane sensor in the cell wall integrity (CWI) pathway. However, a khd1Δ mutation has no detectable phenotype on cell wall synthesis. Here we show that the khd1Δ mutation causes a severe cell lysis when combined with the deletion of the CCR4 gene encoding a cytoplasmic deadenylase. We identified the ROM2 mRNA, encoding a guanine nucleotide exchange factor (GEF) for Rho1, as a target for Khd1 and Ccr4. The ROM2 mRNA level was decreased in the khd1Δ ccr4Δ mutant, and ROM2 overexpression suppressed the cell lysis of the khd1Δ ccr4Δ mutant. We also found that Ccr4 negatively regulates expression of the LRG1 mRNA encoding a GTPase-activating protein (GAP) for Rho1. The LRG1 mRNA level was increased in the ccr4Δ and khd1Δ ccr4Δ mutants, and deletion of LRG1 suppressed the cell lysis of the khd1Δ ccr4Δ mutant. Our results presented here suggest that Khd1 and Ccr4 modulate a signal from Rho1 in the CWI pathway by regulating the expression of RhoGEF and RhoGAP.

Life stage-related differences in susceptibility to acrylamide-induced neural and testicular toxicity.

In order to assess age-dependence of susceptibility to acrylamide (ACR)-induced neural and testicular toxicity, 3- and 7-week-old male SD rats were given ACR at 0, 50, 100, or 200 ppm in the drinking water for 4 weeks, and the nervous and male reproductive systems were examined histopathologically. Testicular genotoxicity was evaluated with the comet assay and the micronucleus (MN) test. Glutathione S-transferase (GST) activity and glutathione (GSH) content in the liver and testis were also measured. In both young and adult animals, neurotoxicity was evident from 100 ppm and increased in proportion to ACR intake per body weight. In the testis, marked degeneration and exfoliation, mainly of spermatids, were observed from 100 ppm limited to young animals. The comet assay revealed ACR to significantly induce DNA damage from 100 ppm in both life stages, while MNs were found only in young rats from 100 ppm. The level of GST activity in the testis of young rats at the end of experiment was significantly lower than that of adult animals, regardless of the ACR treatment. There were no life stage-related differences in GSH contents in the liver and testis. These results suggest that susceptibility to neurotoxicity might not differ between young and adult rats when exposure levels are adjusted for body weight. Regarding testicular toxicity, young animals around puberty proved more susceptible than adult animals, possibly due to their lower level of testicular GST activity than that in adult animals.

Shikonin directly inhibits nitric oxide synthases: possible targets that affect thoracic aorta relaxation response and nitric oxide release from RAW 264.7 macrophages.

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD'(2) value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation-response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 microM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC(50)s, 4 - 7 microM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.

Maternal isobutyl-paraben exposure alters anxiety and passive avoidance test performance in adult male rats.

Isobutyl-paraben (IBP), one of the most widely used preservatives, exhibits estrogenic activity. In this study, we analyzed the effects of maternal IBP treatment on the emotional behavior and learning performance in mature offspring. Pregnant female Sprague-Dawley rats were treated with IBP via a subcutaneous Silastic capsule. Consequently, the offspring were exposed to IBP during gestation through the placentae, and before weaning through the milk. Male and female offspring were tested for emotional behavior in an open field and in an elevated plus maze at five and six weeks old, respectively. IBP-exposed male (but not female) rats spent less time in the open arms of the elevated plus maze. At 11 weeks old, all females were gonadectomized and treated chronically with 17beta-estradiol or cholesterol by Silastic capsules; all males were kept intact. They were tested for learning performance in a passive avoidance test and a Morris water maze. IBP exposure impaired the performance of males in the passive avoidance test. These findings suggest that male rats are more affected by early exposure to IBP than female rats. IBP affects their adult behavior including anxiety and learning abilities.