A novel orexin antagonist from a natural plant was discovered using zebrafish behavioural analysis.

OBJECTIVE: Phenotypic screening is one of the most practical approaches to the identification of mediators of behaviour, since it is difficult to model brain function in vitro, at a cellular level. We used a zebrafish (Danio rerio) behavioural assay to discover novel, natural, neuroactive compounds. MATERIALS AND METHODS: A zebrafish behavioural assay was performed for seven natural compounds, obtained from plants. The behavioural profiles were compared to those of known psychoactive drugs. We characterised a natural compound exhibiting a behaviour profile similar to that of suvorexant, using in silico, in vitro and microarray expression analysis. RESULTS: The behavioural analysis performed in this study classified central nervous system drugs according to their mechanism. Zebrafish treated with a natural compound, 8b-(4'-Hydroxytigloyloxy) costunolide (8b), showed behaviour profiles similar to those of zebrafish treated with suvorexant, a known orexin antagonist. This behavioural assay was validated using in silico and in vitro assays, which revealed that the new compound was a dual orexin receptor antagonist. In addition, transcriptome analysis suggested that 8b might regulate the nuclear factor-κB (NF-κB) related pathway. CONCLUSIONS: We conclude that zebrafish phenotypic screening, combined with in silico assays and gene expression profiling, is a useful strategy to discover and characterize novel therapeutic compounds, including natural products.

Insulin secretion and sensitivity before and after surgical treatment for aldosterone-producing adenoma.

AIM: Primary aldosteronism, which is usually caused by an aldosterone-producing tumour, affects glucose metabolism. The effects of this condition on insulin secretion and insulin sensitivity have remained unclear, however. To gain insight into the influence of primary aldosteronism on glucose tolerance, various parameters related to insulin secretion or insulin sensitivity in patients with an aldosterone-producing tumour were comprehensively analyzed. METHODS: To assess 14 patients with an aldosterone-producing tumour, hyperglycaemic and hyperinsulinaemic-euglycaemic clamp tests as well as oral glucose tolerance tests (OGTTs) were performed before and after tumour excision. Time between presurgical analysis and surgery was 27-559 (194±132) days, and 14-142 (51±38) days between surgery and postsurgical analysis. Various parameters related to insulin secretion or sensitivity as determined by OGTT as well as hyperglycaemic and hyperinsulinaemic-euglycaemic clamp analyses were evaluated. RESULTS: Surgical treatment of tumours ameliorated hypokalaemia and reduced plasma aldosterone levels. First and second phases of insulin secretion during the hyperglycaemic clamp, as well as the insulinogenic index and total insulin secretion measured during OGTT, were also improved after surgery. In addition, the insulin sensitivity index determined during the hyperinsulinaemic-euglycaemic clamp was reduced after surgery. CONCLUSION: Primary aldosteronism impairs insulin secretion.

Restoring PU.1 induces apoptosis and modulates viral transactivation via interferon-stimulated genes in primary effusion lymphoma.

Primary effusion lymphoma (PEL), which is an aggressive subgroup of B-cell lymphoma associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8, is refractory to the standard treatment, and exhibits a poor survival. Although PU.1 is downregulated in PEL, the potential role of its reduction remains to be elucidated. In this investigation, we analyzed the DNA methylation of PU.1 cis-regulatory elements in PEL and the effect of restoring PU.1 on PEL cells. The mRNA level of PU.1 was downregulated in PEL cells. The methylated promoter and enhancer regions of the PU.1 gene were detected in PEL cells. Suppression of cell growth and apoptosis were caused by the restoration of PU.1 in PEL cells. A microarray analysis revealed that interferon-stimulated genes (ISGs) including pro-apoptotic ISGs were strongly increased in BCBL-1 cells after the induction of PU.1. Reporter assays showed that PU.1 transactivated pro-apoptotic ISG promoters, such as the XAF1, OAS1 and TRAIL promoters. Mutations at the PU.1 binding sequences suppressed its transactivation. We confirmed the binding of PU.1 to the XAF1, OAS1 and TRAIL promoters in a chromatin immunoprecipitation assay. PU.1 suppressed ORF57 activation by inducing IRF7. The reinduction of PU.1 reduced formation of ascites and lymphoma cell infiltration of distant organs in PEL xenograft model mice. Collectively, PU.1 has a role in tumor suppression in PEL and its down-regulation is associated with PEL development. Restoring PU.1 with demethylation agents may be a novel therapeutic approach for PEL.

PU.1 acts as tumor suppressor for myeloma cells through direct transcriptional repression of IRF4.

We previously reported that PU.1 is downregulated in the majority of myeloma cell lines and primary myeloma cells of certain myeloma patients, and conditional expression of PU.1 in such myeloma cell lines induced cell cycle arrest and apoptosis. We found downregulation of IRF4 protein in the U266 myeloma cell line following induction of PU.1. Previous studies reported that knockdown of IRF4 in myeloma cell lines induces apoptosis, prompting us to further investigate the role of IRF4 downregulation in PU.1-induced cell cycle arrest and apoptosis in myeloma cells. PU.1 induced downregulation of IRF4 at the protein level, cell cycle arrest and apoptosis in six myeloma cell lines. Chromatin immunoprecipitation (ChIP) revealed that PU.1 directly binds to the IRF4 promoter, whereas a reporter assay showed that PU.1 may suppress IRF4 promoter activity. Stable expression of IRF4 in myeloma cells expressing PU.1 partially rescued the cells from apoptosis induced by PU.1. As it was reported that IRF4 directly binds to the IRF7 promoter and downregulates its expression in activated B cell-like subtype of diffuse large B cell lymphoma cells, we performed ChIP assays and found that IRF4 directly binds the IRF7 promoter in myeloma cells. It is known that IRF7 positively upregulates interferon-ß (IFNß) and induces apoptosis in many cell types. Binding of IRF4 to the IRF7 promoter decreased following PU.1 induction, accompanied by downregulation of IRF4 protein expression. Knockdown of IRF7 protected PU.1-expressing myeloma cells from apoptosis. Furthermore, IFNß, which is a downstream target of IRF7, was upregulated in myeloma cells along with IRF7 after PU.1 induction. Finally, we evaluated the mRNA expression levels of PU.1, IRF4 and IRF7 in primary myeloma cells from patients and found that PU.1 and IRF7 were strongly downregulated in contrast to the high expression levels of IRF4. These data strongly suggest that PU.1-induced apoptosis in myeloma cells is associated with IRF4 downregulation and subsequent IRF7 upregulation.

Association of cigarette smoking with a past history and incidence of herpes zoster in the general Japanese population: the SHEZ Study.

Few studies have examined the impact of cigarette smoking on the risk for herpes zoster. The Shozu Herpes Zoster (SHEZ) Study is a community-based prospective cohort study over 3 years in Japan aiming to clarify the incidence and predictive and immunological factors for herpes zoster. We investigated the associations of smoking status with past history and incidence of herpes zoster. A total of 12 351 participants provided valid information on smoking status and past history of herpes zoster at baseline survey. Smoking status was classified into three categories (current, former, never smoker), and if currently smoking, the number of cigarettes consumed per day was recorded. The participants were under the active surveillance for first-ever incident herpes zoster for 3 years. We used a logistic regression model for the cross-sectional study on the association between smoking status and past history of herpes zoster, and a Cox proportional hazards regression model for the cohort study on the association with risk of incidence. The multivariable adjusted odd ratios (95% CI) of past history of herpes zoster for current vs. never smokers were 0·67 (0·54-0·80) for total subjects, 0·72 (0·56-0·93) for men and 0·65 (0·44-0·96) for women. The multivariable adjusted hazard ratios (95% CI) of incident herpes zoster for current vs. never smokers were 0·52 (0·33-0·81) for total subjects, 0·49 (0·29-0·83) for men and 0·52 (0·19-1·39) for women. Smoking status was inversely associated with the prevalence and incidence of herpes zoster in the general population of men and women aged ⩾50 years.

Functional characterization of a novel GFI1B mutation causing congenital macrothrombocytopenia.

UNLABELLED: Essentials Two groups recently reported GFI1B as a novel causative gene for congenital macrothrombocytopenia. We performed functional analysis of a novel GFI1B mutation and previous mutations. An immunofluorescence analysis of the platelet CD34 expression can be useful as a screening test. Mutant-transduced megakaryocytes produced enlarged proplatelet tips which were reduced in number. SUMMARY: Background GFI1B is an essential transcription factor for megakaryocyte and erythrocyte development. Two groups have recently identified GFI1B as a novel causative gene for congenital macrothrombocytopenia associated with α-granule deficiency. Methods We performed whole exome sequencing and identified a novel GFI1B p.G272fsX274 mutation in a family with macrothrombocytopenia, and a decreased number of platelet α-granules and abnormally shaped red blood cells. p.G272fsX274 and the previous two mutations all predicted disruption of an essential DNA-binding domain in GFI1B. We therefore performed functional studies to characterize the biochemical and biological effects of these three patient-derived mutations. Results An immunofluorescence analysis revealed decreased thrombospondin-1 and increased CD34 expression in platelets from our patient. Consistent with the previous studies, the three patient-derived mutants were unable to repress the expression of the reporter gene and had a dominant-negative effect over wild-type GFI1B. In addition, the three mutations abolished recognition of a consensus-binding site in gel shift assays. Furthermore, transduction of mouse fetal liver-derived megakaryocytes with the three GFI1B mutants resulted in the production of abnormally large proplatelet tips, which were reduced in number. Conclusions Our study provides further proof of concept that GFI1B is an essential protein for the normal development of the megakaryocyte lineage.

Landscape of genetic lesions in 944 patients with myelodysplastic syndromes.

High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model ('Model-1') separating patients into four risk groups ('low', 'intermediate', 'high', 'very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a 'gene-only model' ('Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

PDK1 inhibition is a novel therapeutic target in multiple myeloma.

BACKGROUND: Cancer cells utilise the glycolytic pathway even when adequate oxygen is present, a phenomenon known as the Warburg effect. We examined whether this system is operative in multiple myeloma (MM) cells and whether glycolysis inhibition is a potential therapeutic modality. METHODS: The MM cells were purified from 59 patients using CD138-immunomagnetic beads. The expression levels of genes associated with glycolysis, c-MYC, GLUT1, LDHA, HIF1A and pyruvate dehydrogenase kinase-1 (PDK1) were determined by real-time PCR. Glucose consumption and lactate production by MM cell lines were analysed. Oxamate, an LDH inhibitor, and dichloroacetate (DCA), a PDK1 inhibitor, were employed. Inhibition of PDK1 expression was achieved using a siRNA. RESULTS: High LDHA expression was found to be an indicator of poor prognosis. It was also positively correlated with the expression of PDK1, c-MYC and GLUT1. Greater glucose consumption and lactate production in MM cells was associated with higher LDHA expression. All the glycolysis inhibitors (oxamate, DCA and PDK1 siRNA) induced apoptosis in MM cells. DCA combined with bortezomib showed additive cytotoxic effects. CONCLUSION: The present data suggest that the Warburg effect is operative in MM cells. As PDK1 is not overexpressed in normal tissues, PDK1 inhibition could serve as a novel therapeutic approach.

Assessment of skin test with varicella-zoster virus antigen for predicting the risk of herpes zoster.

The Shozu Herpes Zoster (SHEZ) Study was designed to clarify the incidence of and predictive and immunological factors for herpes zoster in a defined community-based Japanese population. As part of this series, a total of 5683 residents aged ≥50 years received a varicella-zoster virus (VZV) skin test with VZV antigen, and 48 h later, the erythema and oedema were assessed by measuring the longest diameter. The diameters of both the erythema and oedema decreased with the increasing age of the subject. Sixty-three subjects contracted herpes zoster within a year after receiving the VZV skin test. Analysis of the herpes zoster incidence rate vs. the skin test reaction revealed that the shorter the diameter of erythema or oedema, the greater the likelihood of herpes zoster. These results demonstrated that the VZV skin test is an excellent surrogate marker for predicting the risk of herpes zoster.

Colon-specific contractile responses to tetrodotoxin in the isolated mouse gastrointestinal tract.

1 Tetrodotoxin (TTX) is a useful pharmacological tool for distinguishing neural and myogenic responses of isolated visceral organs to drugs. Although TTX does not generally affect smooth muscle tonus, in this study, we have found that TTX causes contraction of the mouse colon. The aim of this study was to characterize this TTX-induced contraction in the mouse gastrointestinal tract. 2 Longitudinal and circular muscle strips from the stomach and small intestine were less sensitive to TTX. However, TTX contracted both smooth muscle strips from the proximal colon and distal colon. 3 Pretreatment with TTX, Nω -nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and apamin inhibited the TTX-induced contraction. L-NAME, ODQ or apamin itself caused contraction in the colon but not in the gastric and small intestinal strips. Region dependency of L-NAME, ODQ and apamin-induced contraction correlated with that of TTX-induced contraction. 4 L-arginine but not D-arginine inhibited contractility of the colonic strips without affecting the contractility of muscle strips from other regions. Sodium nitroprusside caused strong relaxation of the colonic strips. 5 1,1-dimethyl-4-phenylpiperazinium (DMPP) caused relaxation of proximal and distal colons, which was significantly decreased by L-NAME or apamin. 6 In conclusion, among mouse gastrointestinal preparations, TTX induces contraction of colonic strips preferentially through blockade of potent tonic inhibitory neural outflow, which involves nitrergic and apamin-sensitive pathways. Colon-specific responses to L-arginine, L-NAME, ODQ and apamin support the hypothesis that there is a continuous suppression of colonic motility by enteric inhibitory neurons.

PU.1 induces apoptosis in myeloma cells through direct transactivation of TRAIL.

We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.

Characterization of new epidemic strains of influenza B virus by using neutralizing monoclonal antibodies.

During the 1998-1999 influenza season, two distinct influenza B virus Yamagata group strains were isolated from the patients of a private clinic. Each responded differently to monoclonal antibodies (Mabs) 5H4 and 8B3 on staining, and hemagglutination inhibition and neutralizing tests. When the analysis of nucleotide sequences was undertaken, the identity of deduced amino acid sequences of the HA1 region was 94%, which suggested that they derived from different strains. They were termed 5H4-responding strains and 5H4-nonresponding strains, respectively. The analysis of laboratory-induced antigenic variants suggested that the amino acid at position 149 is important to the reactivity to 5H4. This residue was "Arg" in 5H4-responding strains and "Lys" in nonresponding strains. During the 1998-1999 season, a total of 100 influenza B virus strains were isolated and 5H4-responding strains were the major type (94 strains). In the 1999-2000 influenza season, only two influenza B virus strains were isolated. Neither responded to 5H4. However, analysis of the deduced amino acid sequences of the HA1 region suggested that one of the two strains was derived from the 5H4-responding strains of the previous season. The amino acid residue at position 149 was "Lys" in place of "Arg." These observations suggested that 5H4-nonresponding strains will increase in coming seasons.

Regulation of the PU.1 gene by distal elements.

The transcription factor PU.1 (also known as Spi-1) plays a critical role in the development of the myeloid lineages, and myeloid cells derived from PU.1(-/-) animals are blocked at the earliest stage of myeloid differentiation. Expression of the PU.1 gene is tightly regulated during normal hematopoietic development, and dysregulation of PU.1 expression can lead to erythroleukemia. However, relatively little is known about how the PU.1 gene is regulated in vivo. Here it is shown that myeloid cell type-specific expression of PU.1 in stable cell lines and transgenic animals is conferred by a 91-kilobase (kb) murine genomic DNA fragment that consists of the entire PU.1 gene (20 kb) plus approximately 35 kb of upstream and downstream sequences, respectively. To further map the important transcriptional regulatory elements, deoxyribonuclease I hypersensitive site mapping studies revealed at least 3 clusters in the PU.1 gene. A 3.5-kb fragment containing one of these deoxyribonuclease I hypersensitive sites, located -14 kb 5' of the transcriptional start site, conferred myeloid cell type-specific expression in stably transfected cell lines, suggesting that within this region is an element important for myeloid specific expression of PU.1. Further analysis of this myeloid-specific regulatory element will provide insight into the regulation of this key transcriptional regulator and may be useful as a tool for targeting expression to the myeloid lineage.