Genetic analysis of a host determination mechanism of bromoviruses in Arabidopsis thaliana

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.     

Therapeutic effects of a 186Re-complex-conjugated bisphosphonate for the palliation of metastatic bone pain in an animal model.

Previously, based on the concept of bifunctional radiopharmaceuticals, we developed a highly stable (186)Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate] oxorhenium(V) ((186)Re-MAG3-HBP), for the treatment of painful bone metastases. This agent showed a superior biodistribution as a bone-seeking agent in normal mice when compared with (186)Re-1-hydroxyethylidene-1,1-diphosphonate ((186)Re-HEDP). In this study, we evaluated the therapeutic effects of (186)Re-MAG3-HBP using an animal model of bone metastasis. METHODS: The model was prepared by injecting syngeneic MRMT-1 mammary tumor cells into the left tibia of female Sprague-Dawley rats. (186)Re-MAG3-HBP (55.5, 111, or 222 MBq/kg) or (186)Re-HEDP (55.5 MBq/kg) was then administered intravenously 21 d later. To evaluate the therapeutic effects and side effects, tumor size and peripheral blood cell counts were determined. Palliation of bone pain was evaluated by a von Frey filament test. RESULTS: In the rats treated with (186)Re-HEDP, tumor growth was comparable with that in untreated rats. In contrast, when (186)Re-MAG3-HBP was administered, tumor growth was significantly inhibited. Allodynia induced by bone metastasis was attenuated by treatment with (186)Re-MAG3-HBP or (186)Re-HEDP, but (186)Re-MAG3-HBP tended to be more effective. CONCLUSION: These results indicate that (186)Re-MAG3-HBP could be useful as a therapeutic agent for the palliation of metastatic bone pain.     

Intracerebroventricular injection of fusaric acid attenuates the anorexia by glucagon-like peptide-1 in the neonatal chick.

It is known that central injection of glucagon-like peptide-1 (GLP-1) suppresses feeding in rats and chicks, but the systems for GLP-1 are still open with special reference to the chick. The present study was done to determine whether a noradrenergic mechanism contributes to the anorexigenic effect of GLP-1 on the neonatal chick. Central administration of norepinephrine (NE) suppressed food intake with narcolepsy as GLP-1 in chicks. However, in spite of that dopamine (DA) did not affect food intake, coadministration of inhibitor of dopamine-beta-hydroxylase (DBH), fusaric acid (FA), attenuated the suppressive effect of GLP-1 on feeding behavior. It is suggested that there may be the interactive relationships between GLP-1 and noradrenergic system in the neonatal chick.     

Design and baseline characteristics of an observational study in Japanese patients with hypertension: Japan Hypertension Evaluation with Angiotensin II Antagonist Losartan Therapy (J-HEALTH).

The Japan Hypertension Evaluation with Angiotensin II Antagonist Losartan Therapy (J-HEALTH) study is a nationwide, prospective, multicenter observational study that was designed to enroll hypertensive Japanese patients (>30,000 subjects). The patients in this study received treatment with open-label losartan, an angiotensin II receptor antagonist, for a maximum of 5 years. This report summarizes the study protocol and the baseline characteristics of the patients. Between June 2000 and May 2002, patients were screened in all 47 prefectures around Japan. Among the 31,515 patients screened, 31,048 patients were enrolled in this study and treated with losartan at a daily dose of 25-50 mg. These patients were 62.4 +/- 12.1 years old (mean +/- SD) and the mean clinic systolic/diastolic blood pressure (BP) values were 165.3 +/- 17.3/94.3 +/- 11.7 mmHg (mean +/- SD). The complications of hyperlipidemia, diabetes mellitus, cardiovascular disease, and cerebrovascular disease were also present in 38.5%, 13.1%, 8.0%, and 4.4% of patients, respectively. Regarding the World Health Organization classification, grade 2 hypertension was most frequent in this patient cohort. Nearly 10,000 patients agreed to perform home BP monitoring and report details regarding their lifestyles at baseline. Among the patients, 4.2% had white coat hypertension at the baseline. The J-HEALTH study is expected to provide valuable information about the significance of clinic and home BP control and home BP monitoring for the management of hypertension in Japanese patients.     

Identification of differentially expressed genes in hepatic HepG2 cells treated with acetaminophen using suppression subtractive hybridization

Acetaminophen (APAP) is widely used for the treatment of pain and fever. Although it is safe at therapeutic doses, APAP is toxic at higher doses and can cause severe damage to the liver. To clarify the mechanism of APAP-related liver damage, we attempted the identification of the differential gene expression in response to APAP treatment in hepatic HepG2 cells. In the present study, we used the technique of suppression subtractive hybridization (SSH) for the identification of the differentially expressed genes between untreated and treated cells and identified 14 candidate genes showing increased expression in response to APAP treatment. RT-PCR and real-time RT-PCR analysis confirmed that the expression of two genes was increased within 24 h following APAP treatment. Among them, only lysyl hydroxylase 2 expression was increased in a time- and dose-dependent manner. Furthermore, the expression of lysyl hydroxylase 2 was shown to be increased in the livers of APAP-treated mice compared to untreated controls. The increased expression of lysyl hydroxylase 2 was also observed when the cells were exposed to other hepatotoxins, ethanol and isoniazid. Since lysyl hydroxylase 2 is known to be a key enzyme of liver fibrosis, the increased expression of lysyl hydroxylase 2 may be involved in hepatotoxins-related liver fibrosis.     

Clinicopathological and molecular alterations in early gastric cancers with the microsatellite instability‐high phenotype

The relevance of the clinicopathological and molecular features of early gastric cancers (EGCs) having the microsatellite instability (MSI)‐high phenotype has not been clearly defined in sporadic gastric carcinogenesis. Here, we examined the clinicopathological and molecular characteristics of EGC according to MSI status in 330 patients with EGC (intestinal‐type adenocarcinoma). Tumors were classified as MSI‐high (45 cases), MSI‐low (9 cases), or microsatellite stable (MSS; 276 cases). The specimens were examined using a combination of polymerase chain reaction (PCR)‐microsatellite assays and PCR‐pyrosequencing to detect chromosomal allelic imbalances in multiple cancer‐related chromosomal loci, MSI, gene mutations (KRAS and BRAF) and methylation status [high methylation epigenome (HME), intermediate methylation epigenome and low methylation epigenome]. In addition, the expression levels of various target proteins were examined using immunohistochemistry. Interestingly, EGC with the MSI phenotype showed distinct papillary features. The expression of gastric mucin was more frequent in EGC with the MSI phenotype, while p53 overexpression was common in EGCs, irrespective of MSI status. The frequency of HME was significantly higher in EGCs with the MSI phenotype than in EGCs with the MSS phenotype. Although there was a low frequency of allelic imbalance in EGCs with the MSI phenotype, some markers of allelic imbalance were more frequently detected in EGCs with the MSI‐high phenotype than in EGCs with the MSS phenotype. KRAS and BRAF mutations were rare in EGCs. Thus, the MSI phenotype in EGC is a major precursor lesion in gastric cancer and is characterized by distinct clinicopathological and molecular features.     

Pharmacokinetics of arsenic species in Japanese patients with relapsed or refractory acute promyelocytic leukemia treated with arsenic trioxide

Purpose To investigate the pharmacokinetics of arsenic species in Japanese patients with relapsed or refractory acute promyelocytic leukemia (APL) treated with arsenic trioxide (ATO) at a daily dose of 0.15 mg/kg. Methods Inorganic arsenic (As^III and As^V) and the major metabolites monomethylarsonic acid (MAA^V) and dimethylarsinic acid (DMAA^V) in plasma and urine collected from 12 Japanese patients were quantified by HPLC/ICP-MS. Results The plasma concentrations of As^III and As^V on day 1 reached the similar Cmax (12.4 ± 8.4 and 10.2 ± 3.9 ng/ml) immediately after completion of administration followed by a biphasic elimination. The AUC_0–∞ of As^V was about twice that of As^III. The appearance of methylated metabolites in the blood was delayed. During the repeated administration, the plasma concentrations of inorganic arsenic reached the steady state. In contrast, the MAA^V and DMAA^V concentrations increased in relation to increased administration frequency. The mean total arsenic excretion rate including inorganic arsenic and methylated arsenic was about 20% of daily dose on day1 and remained at about 60% of daily dose during week 1–4. Conclusions This study demonstrates that ATO is metabolized when administered intravenously to APL patients and methylated metabolites are promptly eliminated from the blood and excreted into urine after completion of administration, indicating no measurable accumulation of ATO in the blood. This study was partly supported by a Grant-in-aid for Cancer Research (no. 9–2) of the Ministry of Health and Welfare.     

SERPINI1 regulates epithelial–mesenchymal transition in an orthotopic implantation model of colorectal cancer

An increasingly accepted concept is that the progression of colorectal cancer is accompanied by epithelial–mesenchymal transition (EMT). In our study, in order to characterize the properties of EMT in 16 colorectal cancer cell lines, the cells were first orthotopically implanted into nude mice, and the tumors in vivo, as well as cells cultured in vitro, were immunostained for EMT markers. The immunostaining revealed that seven of the cells had an epithelial phenotype with a high expression of E‐cadherin, whereas other cells showed opposite patterns, such as a high expression of vimentin (CX‐1, COLO205, CloneA, HCT116, and SW48). Among the cells expressing vimentin, some expressed vimentin in the orthotopic tumors but not in the cultured cells (SW480, SW620, and COLO320). We evaluated these findings in combination with microarray analyses, and selected five genes: CHST11, SERPINI1, AGR2, FBP1, and FOXA1. Next, we downregulated the expression of SERPINI1 with siRNA in the cells, the results of which showed reverse‐EMT changes at the protein level and in the cellular morphology. Along with immunohistochemical analyses, we confirmed the effect of the intracellular and secreted SERPINI1 protein of SW620 cells, which supported the importance of SERPINI1 in EMT. The development of therapeutic strategies targeting EMT is ongoing, including methods targeting the transforming growth factor‐β signaling pathway as well as the Wnt pathway. SERPINI1 is an important regulator of EMT. Our findings help to elucidate the signaling pathways of EMT, hopefully clarifying therapeutic pathways as well.     

Intracerebroventricular administration of mouse leptin does not reduce food intake in the chicken

Recently, it has been suggested that leptin plays an important role in regulation of food intake and metabolism in rats and mice, however, the effect of central administration of leptin on food intake in chicks has not been reported. We have investigated the anorexigenic effect of leptin administered by intracerebroventricular (i.c.v.) injection in chicks using mouse leptin, which shows 97% homology to chicken leptin. Three experiments were conducted. After being deprived of food for 3 h, male broiler chicks were administered leptin by i.c.v. injection at dose levels of 0, 0.2, 1.0 and 5.0 μg (Experiment 1) or 0, 2.5 and 5.0 μg (Experiment 2). The birds were allowed free access to the diet for 2 h (Experiment 1) and 24 h (Experiment 2) after treatment. Male Single Comb White Leghorn chicks were used in Experiment 3 and were treated in the same manner as in Experiment 1. In all experiments, central administration of mouse leptin did not influence food intake in the time periods examined. It appears that either mouse leptin does not bind to the chicken leptin receptor or in the chicken brain the leptin receptor may be absent.