Isolation of a reduced form of cyanidin 3-O-ß-D-glucoside from immature black soybean (Glycine max (L.) Merr.) and its reducing properties.

5,7,3',4'-Tetrahydroxyflav-2-en-3-ol 3-O-ß-D-glucoside was isolated from the seed coats of immature black soybeans (Glycine max (L.) Merr.). This compound is a reduced form of cyanidin 3-O-ß-D-glucoside (cyanidin 3-G) which was obtained by reaction with hydrochloric acid. The molecule has reducing activity for a tetrazolium derivative (WST-1) in the presence of 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) in a similar manner to NADH. The seed coats of immature black soybeans also contain epicatechin as a major constituent, while cyanidin 3-G and procyanidin B2 are present at lower concentrations. Immature brown soybeans did not contain 5,7,3',4'-tetrahydroxyflav-2-en-3-ol 3-O-ß-D-glucoside, but did contain both epicatechin and procyanidin B2. Immature yellow soybeans contained none of them.

A hair growth-promoting effect of Chinese black tea extract in mice.

Chinese black tea extract (CBTE) fermented with Aspergillus sp. significantly promoted hair growth after 2 weeks of topical application in shaved 6 week-old male C3H/He mice. The hair growth-promoting effect of CBTE was potentiated synergistically by capsaicin, which has no effect on hair growth by itself. CBTE displayed an affinity for estrogen receptor (ER)α, with an IC50 value of 74.8 µg/mL. This effect of CBTE might be mediated by the ERs, since a similar effect induced by orally administered soy isoflavone, a mixture of ERs ligands, has been reported to be synergistically potentiated by capsaicin.

Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension.

Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.

OAZ-t/OAZ3 is essential for rigid connection of sperm tails to heads in mouse.

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t-disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines.

A redox-silent analogue of tocotrienol acts as a potential cytotoxic agent against human mesothelioma cells.

AIMS: Malignant mesothelioma is an aggressive cancer with no effective treatment options. A redox-silent analogue of alpha-tocotrienol, 6-O-carboxypropyl-alpha-tocotrienol (T3E) is a new potential anti-carcinogenic agent with less toxic effect on non-tumorigenic cells. Here, we evaluated the effect of T3E on killing of chemoresistant mesothelioma cell (H28). MAIN METHODS: The cytotoxic effect of T3E was evaluated by a WST-1 assay, and cell cycle and apoptosis analysis were done by FACS. Each signal molecule's activity was determined by protein array and immunoblot analysis. KEY FINDINGS: T3E effectively inhibited H28 cell growth at practical pharmacological concentrations (10-20 muM) without any effect on non-tumorigenic mesothelial cell (Met-5A). Inhibition of H28 cell growth by T3E mediated through G2/M arrest in cell cycle and induction of apoptosis. Protein array and immunoblot analyses revealed that T3E inhibited the activation of epidermal growth factor receptor (EGFR) via the inactivation of the Src family of protein tyrosine kinases (Src). However, the blockade of the EGFR signaling was not associated with the T3E-dependent H28 cell growth control. In addition to Src inactivation, T3E inhibited signal transduction and activation of transcription Stat3. A combination of an Src inhibitor, PP2, and a Stat3 inhibitor, AG490, induced G2/M arrest and enhanced apoptosis compared with PP2 alone. These results suggest that T3E suppresses H28 cell growth via the inhibition of Src activation and Src-independent Stat3 activation. SIGNIFICANCE: T3E can be a new effective therapeutic agent against chemoresistant mesothelioma cells.

LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein.

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments co-migrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin-1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co-precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein-LIS1 complex on transportable MTs, which is a possibility supported by our data.

Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation.

Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

A redox-silent analogue of tocotrienol inhibits hypoxic adaptation of lung cancer cells.

We have previously reported that a redox-silent analogue of alpha-tocotrienol (T3), 6-O-carboxypropyl-alpha-tocotrienol (T3E) shows more potential anti-carcinogenic property than T3 in a lung cancer cell (A549 cell). However, the mechanisms by which T3E exerts its potential anti-carcinogenic effect is still unclear. As tumor malignancy is associated with hypoxia adaptation, in this study, we examined whether T3E could suppress survival and invasion in A549 cells under hypoxia. Hypoxia treatment drastically-induced activation of the protein tyrosine kinase, Src, and its regulated signaling required for hypoxia adaptation of A549 tumor cells. The survival and invasion capacity of the tumor cells under hypoxia was suppressed by T3E via the inactivation of Src. More specifically, T3E-dependent inhibition of Src-induced Akt activation contributed to suppression of cell survival under hypoxia, and the reduction of fibrinolytic factors such as plasminogen activator-1(PAI-1) via the decrease of hypoxia-inducible factor-2alpha by T3E led to inhibition of hypoxic invasion. Overall these results suggest that T3E suppresses hypoxia adaptation of A549 cells by the inhibition in hypoxia-induced activation of Src signaling.

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment.

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.

N-acetylcysteine and S-methylcysteine inhibit MeIQx rat hepatocarcinogenesis in the post-initiation stage.

N-acetylcysteine (NAC) and S-methylcysteine (SMC), water soluble organosulfur compounds contained in garlic, were evaluated for chemoprevention of hepatocarcinogenesis after 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) initiation in rats. Intergastric treatment with NAC or SMC five times a week resulted in decreased numbers and areas of preneoplastic, glutathione S-transferase placental form (GST-P) positive foci of the liver in a dose-dependent manner. Moreover, cell proliferation was reduced in GST-P positive foci by NAC and SMC. Insulin-like growth factor I (IGF-I) and inducible nitric oxide synthase (iNOS) mRNA expressions were found downregulated in the liver by NAC. The studies indicate that NAC can serve as a chemopreventive agent for rat hepatocarcinogenesis induced by MeIQx by reducing cell proliferation, which may involve IGF-I and iNOS downregulation.

Recruitment of katanin p60 by phosphorylated NDEL1, an LIS1 interacting protein, is essential for mitotic cell division and neuronal migration.

LIS1 is mutated in the human neuronal migration defect lissencephaly and along with NDEL1 (formerly NUDEL) participates in the regulation of cytoplasmic dynein function during neuronal development. Targeted disruption of Ndel1 suggested that NDEL1 could have other molecular targets that regulate microtubule organization for proper neuronal migration. To further understanding the molecular mechanism of LIS1 and lissencephaly, we identified the katanin p60 microtubule-severing protein as an additional molecular target of NDEL1. We demonstrate that phosphorylation of NDEL1 by Cdk5 facilitates interaction between NDEL1 and p60, suggesting that P-NDEL1 regulates the distribution of katanin p60. Abnormal accumulation of p60 in nucleus of Ndel1 null mutants supports an essential role of NDEL1 in p60 regulation. Complete loss of NDEL1 or expression of dominant negative mutants of p60 in migrating neurons results in defective migration and elongation of nuclear-centrosomal distance. Our results suggest that NDEL1 is essential for mitotic cell division and neuronal migration not only via regulation of cytoplasmic dynein function but also by modulation of katanin p60 localization and function.

Peroxisome proliferator-activated receptor delta as a molecular target to regulate lung cancer cell growth.

It has been assumed that prostaglandin (PG)I2 signaling contributes to the negative growth control of lung cancer cells; however, the mechanism remains unresolved. PGI2 functions through a cell surface G protein-coupled receptor (prostaglandin I2-binding receptor, IP) and also exerts an effect by interacting with a nuclear hormone receptor, peroxisome proliferator-activated receptor delta (PPARdelta). We found that PPARdelta was a key molecule of PGI2 signaling to give negative growth control of lung cancer cells (A549), using carbarprostacyclin, a PGI2 agonist for IP and PPARdelta, and L-165041, a PPARdelta agonist. Furthermore, PPARdelta-induced cell growth control was reinforced by the inhibition of cyclooxygenase. These results suggest that PPARdelta activation under the suppression of PG synthesis is important to regulate lung cancer cell growth.

Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxypropyl-alpha-tocotrienol, a redox-silent derivative of alpha-tocotrienol.

Tocotrienols are one of the most potent anticancer agents of all natural compounds and the anticancer property may be related to the inactivation of Ras family molecules. The anticancer potential of tocotrienols, however, is weakened due to its short elimination half life in vivo. To overcome the disadvantage and reinforce the anticancer activity in tocotrienols, we synthesized a redox-silent analogue of alpha-tocotrienol (T3), 6-O-carboxypropyl-alpha-tocotrienol (T3E). We estimated the possibility of T3E as a new anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras gene. T3E showed cytotoxicity against A549 cells, a human lung adenocarcinoma cell line with a ras gene mutation, in a dose-dependent manner (0-40 microM), whereas T3 and a redox-silent analogue of alpha-tocopherol (T), 6-O-carboxypropyl-alpha-tocopherol (TE), showed much less cytotoxicity in cells within 40 microM. T3E cytotoxicity was based on the accumulation of cells in the G1-phase of the cell-cycle and the subsequent induction of apoptosis. Similar to this event, 24-hr treatment of A549 cells with 40 microM T3E caused the inhibition of Ras farnesylation, and a marked decrease in the levels of cyclin D required for G1/S progression in the cell-cycle and Bcl-xL, a key anti-apoptotic molecule. Moreover, the T3E-dependent inhibition of RhoA geranyl-geranylation is an inducing factor for the occurrence of apoptosis in A549 cells. Our results suggest that T3E suppresses Ras and RhoA prenylation, leading to negative growth control against A549 cells. In conclusion, a redox-silent analogue of T3, T3E may be a new candidate as an anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras genes.

A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene.

We have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2-3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5-1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to "mutation" or "variation" of pseudogenes.

p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection.

BACKGROUND: Epigenetic alteration through methylation is one of the most important steps in carcinogenesis. However, the relation between hepatitis virus infection and epigenetic alterations is poorly understood. METHODS: Sixteen patients without hepatitis B virus (HBV) and hepatitis C virus (HCV) and 35 patients with HBV or HCV who underwent liver resection for hepatocellular carcinoma (HCC) were studied. Mutation of p53 was detected by direct sequencing. Methylation status of p16 was evaluated in tumor and noncancerous liver tissues by methylation-specific polymerase chain reaction. RESULTS: In HCC without HBV and HCV, p53 mutations were detected in 5 (31%) of 16 HCCs. Methylation of p16 promoter was detected in 2 (25%) of 8 moderately differentiated HCCs, 6 (75%) of 8 poorly differentiated HCCs, and none of 16 noncancerous tissue specimens. In HCC with HBV or HCV, p53 mutations were detected in 8 (23%) of 35 HCCs. Methylation of p16 promoter was detected in 2 (100%) of 2 well-differentiated HCCs, 13 (76%) of 17 moderately differentiated HCCs, 12 (75%) of 16 poorly differentiated HCCs, and 9 (26%) of 35 noncancerous liver tissue specimens. CONCLUSIONS: Our results suggest that hepatitis viruses might induce methylation of p16 promoter in liver with chronic inflammation, before appearance of HCC.

Identification of a new target molecule for a cascade therapy of polycystic kidney.

Autosomal dominant polycystic kidney disease is a systemic disorder that primary affects the kidney which is characterized by the formation of fluid-filled cysts in both kidneys that leads to progressive renal failure. Mutated genes, polycystin-1 and polycystin-2, are identified, and evidence has emerged that polycystins are ion channels or regulators of ion channels. In spite of extensive characterization of polycystins, how polycystin channel signaling may be involved in cyst formation in ADPKD is still unclear. We found a mutant mouse which exhibits polycystic kidney and bone deformity in the course of making a transgenic mouse carrying the Drosophila sex-lethal gene. We identified a mutated gene Makorin1 by positional cloning. Makorin1 carries a typical RING-finger motif, suggesting that Makorin1 belongs to ubiquitinase E3 family. Makorin1 would open a new avenue to understand pathogenesis of polycystic kidney, and become a new therapeutic target of polycystic kidney.

Detection of serum hepatitis B virus DNA by real-time quantitative polymerase chain reaction (TaqMan PCR) during lamivudine treatment: comparison with three other assays.

Monitoring of hepatitis B virus (HBV) levels in serum plays an important role in the management of chronic hepatitis B in patients receiving lamivudine. We evaluated the usefulness of real-time quantitative polymerase chain reaction (TaqMan PCR) for the measurement of HBV DNA. The subjects were 22 patients with chronic hepatitis B treated with lamivudine for 4-12 months. HBV DNA was measured by TaqMan PCR. For comparison, HBV DNA was also measured in 88 sera by branched DNA (bDNA) assay, transcription-mediated amplification (TMA) assay, and Amplicor monitor test. Correlation was significant between the results of TaqMan PCR and those of the three other assays (r=0.630, 0.681, and 0.715, respectively; P<0.05). Of the 22 patients, HBV DNA was beneath the detection limit at the start of therapy in 4 (18%) on the bDNA assay, 3 (14%) on the TMA assay, 2 (9%) on the Amplicor test, and 0 (0%) on TaqMan PCR. Of the 19 patients for whom sera were available at 12 weeks of therapy, HBV DNA was not detected in 16 (84%) on the bDNA assay, 12 (63%) on the TMA assay, 6 (32%) on the Amplicor test, and 2 (11%) on TaqMan PCR. Tyrosine-methionine-aspartate-aspartate (YMDD) variants emerged in three patients; TaqMan PCR detected HBV DNA throughout treatment and revealed significantly increased viral loads before biochemical breakthrough. We conclude that monitoring of HBV by TaqMan PCR is useful for evaluating response to lamivudine treatment and for early detection of drug-resistant variants.

Association of HLA alleles with response (especially biochemical response) to interferon therapy in Japanese patients with chronic hepatitis C.

The response of chronic hepatitis C to interferon (IFN) treatment is classified as complete response (CR), biochemical response (BR), or no response (NR). Several studies have found no difference in prevention of hepatocellular carcinoma by IFN therapy between patients with CR and those with BR. We investigated whether specific human leukocyte antigen (HLA) alleles were associated with response to IFN, especially BR, in 138 patients with chronic hepatitis C. Comparing patients with and without CR, male, a low viral titer, genotype 2a or 2b, HLA-B55, and HLA-DRB1-0803 were more common in the group with CR. Multivariate analysis showed that age (adjusted odds ratio [OR], 0.95 by every year [95% confidence interval [CI] 0.90 - 0.99], p = 0.028), genotype 2a or 2b (5.21 [95% CI 1.63 - 16.6], p = 0.005), and low viral titer (8.58 (2.66 - 27.7), p < 0.001) were associated with CR. Comparing patients with BR and NR, the pretreatment alanine aminotransferase (ALT) level was lower in the BR group (p < 0.001). Both HLA-B7 and HLA-DRB1-0101 were more common in this group (p = 0.002). As the alleles HLA-B7 and HLA-DRB1-0101 were in linkage disequilibrium, the HLA-B7-DRB1-0101 haplotype may be associated with BR. Multivariate analysis indicated that a low ALT level (0.98 by every 1 IU/L [95% CI 0.98 - 0.99], p = 0.001) and HLA-B7-DRB1-0101 haplotype (32.3 [95% CI 1.50 - 693.1], p = 0.026) contributed significantly to BR. This study suggested that host HLA expression, but not viral factors, can influence BR.

Post-initiation inhibition of MeIQx hepatocarcinogenesis in rats by cysteine.

Rats were administered cysteine at a dose of 100 mg/kg b.w. 5 times per week after 2-amino-3, 8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) treatment. Significant decrease in numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci, putative preneoplastic lesions, and silver-stained nucleolar organizer regions were evident in the livers of rats treated with cysteine after MeIQx treatment. Morever, post-initiation stage cysteine treatment resulted in decreased hepatic insulin-like growth factor (IGF)-I mRNA expression. Thus post-initiation cysteine treatment may exert chemopreventive effect on MeIQx hepatocarcinogenesis.

Peroxisome proliferator-activated receptor gamma activation induces cell cycle arrest via the p53-independent pathway in human anaplastic thyroid cancer cells.

Anaplastic thyroid carcinoma is one of the most aggressive human malignancies. Outcomes of intensive multimodal therapy have been far from satisfactory. Furthermore, p53 gene dysfunction, often found in this type of cancer, is known to impair the efficacy of the therapeutic agents. Specific ligands for peroxisome proliferator activated receptor gamma (PPAR-gamma) induce growth suppression in some tumor cells. In this study, we investigated the role of PPAR-gamma in anaplastic thyroid cancer cell lines (OCUT-1, ACT-1). PPAR-gamma was expressed and functional in both cell lines. Activation of PPAR-gamma with its specific ligands, troglitazone and 15-deoxy-delta 12,14-prostaglandin J2, inhibited cell growth in a dose-dependent manner through inducing G1 cell cycle arrest. P53 protein expression differed in OCUT-1 and in ACT-1, though the levels stayed constant irrespective of ligand exposure in both cell lines. In contrast, p21 and p27 proteins were induced in a dose-dependent manner in both situations. This study showed that PPAR-gamma ligands were able to induce growth suppression in anaplastic thyroid cancer cells via a p53-independent, but p21- and p27-dependent cytostatic pathway. These tumor-suppressive effects of PPAR-gamma may provide a novel approach to the treatment of anaplastic thyroid cancer.