Identification of a chemical inhibitor for nuclear speckle formation: implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing.

Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A)(+) RNAs comprising mRNAs and poly (A)(+) non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A)(+) RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

Taurine as a marker for the identification of natural Calculus Bovis and its substitutes.

Calculus Bovis (C. Bovis) is a commonly used animal-derived therapeutic preparation. To meet the increasing clinical demand for the preparation, two artificial substitutes for Bos Taurus have been introduced in China: artificial C. Bovis and in vitro cultured C. Bovis. However, information on their efficacy and safety is inadequate. Therefore, we investigated the biological differences between the commonly used natural preparation and its two substitutes, with the aim of not only identifying the differences but also providing a procedure to distinguish between the different preparations.In the study, we prepared 9 natural C. Bovis, 2 artificial C. Bovis, and 2 in vitro cultured C. Bovis preparations for evaluation. Differences were noted between the three preparations relative to their effect on viability of cardiac fibroblasts from 1-day-old Wistar rats. Although natural C. Bovis had no effect on cell viability, 1-h treatment of the cells with 0.25 mg/ml of the substitutes significantly reduced cell viability, as detected by the MTS assay. Based on liquid chromatography and inductively coupled plasma mass spectrometry, the preparations also differed in composition. Indeed, the substitutes contained more taurine, cholic acid, iron, magnesium, and calcium than the natural preparations. They also differed spectroscopically.The present results reveal significant biological differences between natural C. Bovis and two of its substitutes. Since the substitutes appear to contain more taurine, cholic acid, and elements, these constituents may serve as markers to distinguish between natural C. Bovis and its substitutes.

Quality and safety issues related to traditional animal medicine: role of taurine.

BACKGROUND: Calculus Bovis (:C.Bovis) is one of the most precious and commonly-used medicinal materials in Japan and China. As the natural occurrence is very rare, a source of supply for C. Bovis is far behind the actual need and great efforts have been taken for some substitutes of natural C. Bovis. Unfortunately, very little information is available on the quality and/or clinical efficacy of medication based on C. Bovis. To ensure sustainable use of traditional therapeutic agents derived from C. Bovis, we felt that several issues needed to be addressed: 1) the source of the C. Bovis materials and quality control; 2) the role of taurine in the efficacy of C. Bovis. METHODS: Nine samples of natural C. Bovis and its substitutes were collected. ICP-MS was used for elemental analysis and the characterization was performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) as multivariate approaches. The efficacy of C. Bovis was evaluated for morphology, viability and beating pattern on cultured cardiac myocytes and/or fibroblasts. RESULTS: PCA and multi-elemental focus was effective in discriminating C. Bovis samples derived from different habitats. A satisfactory classification using SIMCA was obtained among Australia C. Bovis, other habitats and the substitutes. Australian samples had better batch uniformity than other habitats and were composed of fewer elements. We have used Australian C. Bovis for assessment on its bioactive compounds. Rat cardiac cells incubated with C. Bovis extract (0.01-0.1 mg/ml) maintained normal morphology, viability and beating pattern. Cardiac myocytes and fibroblasts treated for 48 h with CA (0.5 mM) or DCA (0.1 mM) caused cell injury, as reflected by changes in appearance and a reduction of viability detected by the MTS assay. In cardiomyocytes, 0.5 h exposure of CA (0.5 mM) markedly decreased the velocity ratio of beating, whereas the simultaneous addition of 1 mM taurine largely prevented the decrease. CONCLUSIONS: The multi-elemental focus provided some references for the quality control and the efficacy of C. Bovis. Taurine partly attenuated the harmful actions of bile acids. It is plausible that the relationship between taurine and the bile acids contributes to therapeutic effect of C. Bovis.

Persistent BDNF exon I-IX mRNA expression following the withdrawal of neuronal activity in neurons.

It is still unclear whether an active state of transcription once established in chromatin persists in neurons. Here, we focused on BDNF exon I-IX mRNA expression because of its marked induction upon the treatment of rat cortical neurons with trichostatin A, suggesting strong repression of the expression through histone deacetylase activity. Acetylation of histones H3 and H4 in promoter-I of the BDNF gene (BDNF-PI) was induced by membrane depolarization time- and dose-dependently, corresponding with the increase in mRNA expression. Following withdrawal of the depolarization, the mRNA level remained elevated for at least 6h, the persistence of which depended upon the strength of depolarization, whereas the BDNF exon IV-IX expression did not. The acetylation of histones was also maintained with BDNF-PI. Thus, BDNF exon I-IX mRNA expression remained increased after depolarization was withdrawn, suggesting that once activated, the BDNF-PI transcription persists due to chromatin remodeling.

Tool from traditional medicines is useful for health-medication: Bezoar Bovis and taurine.

Bezoar Bovis (BB:dried cattle gallbladder stones) has been used empirically in Asia for over 3000 years to treat heart and liver disorders. Yet its therapeutic potential remains unexplored by Western researchers. The aim of this study has been to clarify the actions of BB on cultured cardiomyocytes and to identify its active component(s). BB is a component of 98.7% of the Japanese over the counter (OTC) cardioactive drugs. The water-extract of BB exhibits protection action against arrhythmias produced by low Ca2+ and high Ca2+ in the medium. On the other hand, the Ca(2+)-antagonist, verapamil, did not suppress arrhythmias that developed in cell culture. Rather, it aggravated the beating status of the cardiomyocytes. The major constituents of the BB extract are bile salts (cholate, deoxycholate, taurocholate) and amino acids (taurine, cysteine, leucine, isoleucine). Most cells incubated with bile salts developed morphological damage. However, one of the major constituents of the BB extract, taurine, was effective in protecting against the abnormal beating pattern induced by high Ca2+. Since beta-alanine, an inhibitor of taurine transport, antagonized the protective effects of both BB and taurine, it is likely that the effect of BB is partly mediated by taurine.

Molecular cloning and functional characterization of porcine nucleotide-binding oligomerization domain-2 (NOD2).

The nucleotide-oligomerization domain (NOD) 2 is an important molecule involved in host defense. In this study, we report the cloning and characterization of porcine NOD2 (poNOD2) cDNA. The open reading frame of poNOD2 contains 3042 bp which encode 1013 amino acid residues. The putative poNOD2 protein shares higher level of homology with human counterpart (81.6% amino acid identity) than the mouse protein (76.6% amino acid identity). In order to determine the function of poNOD2, we established human embryonic kidney (HEK) 293 cells transfected to express poNOD2 cDNA. We found that poNOD2 was expressed not only in the cytoplasm but also in the inner side of the plasma membrane of HEK293 cells. HEK293 cells expressing poNOD2 responded to muramyl dipeptide (MDP) by activation of the nuclear factor kappa B (NF-kappaB). Quantitative real-time PCR revealed that poNOD2 mRNA was expressed by a number of tissues isolated from adult and newborn swine such as esophagus, duodenum, jejunum, ileum, ileal Peyer's patches (Pps), colon, spleen, and mesenteric lymph nodes (MLNs). In the newborn swine, the expression of poNOD2 mRNA was detected at higher levels in MLNs and spleen as compared to other tissues. In the adult swine, the highest expression was observed in ileal Pps. Furthermore, Toll-like receptor (TLR) and NOD2 ligands as well as immunobiotic lactic acid bacteria (LAB) enhanced the expression of NOD2 in gut-associated lymphoid tissues (GALT) in adult and newborn swine. Our results implicate NOD2 as an important immunoregulator in the swine intestinal immunity.

Robust stimulation of TrkB induces delayed increases in BDNF and Arc mRNA expressions in cultured rat cortical neurons via distinct mechanisms.

In cultures of rat cortical neurons, we found that stimulation of tyrosine receptor kinase B (TrkB) with brain-derived neurotrophic factor (BDNF) induced a biphasic expression of BDNF exon IV-IX mRNA, which became obvious 1-3 h (primary induction) and 24-72 h (delayed induction) after the stimulation, and characterized the delayed induction in relation to the mRNA expression of activity-regulated cytoskeleton-associated protein (Arc). Withdrawal of BDNF from the medium after stimulation for 3 h allowed the delayed induction, which was caused at the transcriptional level and dependent upon the initial contact between exogenously added BDNF and TrkB, the effect of which was time- and dose-dependent. The primary induction was controlled by the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) whereas the secondary induction by the calcium (Ca(2+)) signaling pathway. The enhanced Arc or Zif268 mRNA expression was controlled by activation of the ERK/MAPK pathway, both of which were repressed by blocking the binding of endogenously synthesized BDNF to TrkB. Thus, robust stimulation of TrkB autonomously induces delayed BDNF mRNA expression in an activity-dependent manner in rat cortical neurons, resulting in the stimulation of Arc mRNA expression through endogenously synthesized BDNF, the process being orchestrated by the Ca(2+) and ERK/MAPK signaling pathways.

[Effects of nutrition education for residents on intake of lipid-related nutrients].

OBJECTIVES: Health education for residents is now common, but only a few studies of its effects have been made. The purpose of this study was to investigate the effects of nutrition education for residents on intake of lipid-related nutrients. METHODS: A total of 79 females (40-64 years) who underwent a health examination for residents in a town, Nara Prefecture and were found to have total serum cholesterol levels between 220 mg/dl and 300 mg/dl were divided into two groups. In the first group, nutrition education was performed during the first 24 weeks and no education was performed during the second 24 weeks as the self-control period. In the second group, no education was given during the first 24 weeks as the waiting period but was performed during the second 24 weeks. During the education period, dietary intervention for individual subjects was performed 3 times at intervals of 8 weeks by trained dietitians. The intake of nutrients was estimated by the food frequency questionnaire developed by Ueshima and Okayama, and changes in the intake of nutrients adjusted for total energy were used for evaluation of the effects of the education. RESULTS: In the first group of 42 subjects, three discontinued during the education period and two during the self-control period, and in the second group of the 37 subjects, six discontinued during the waiting period and three during the education period. At the end of the education period, for the total of 67 subjects (39 and 28 in the first and second groups, respectively), the total energy adjusted intake of lipid, cholesterol and saturated fatty acid were significantly lower and the PS ratio was significantly higher than in the second group during the waiting period. During the self-control period after the education, the adjusted intake of lipid-related nutrients remained unchanged in the 37 subjects of the first group who had been given the nutrition education in the first 24 weeks, and it was significantly lower at the end of the 48-week test period than at the baseline examination. The percentage of the subjects showing a desirable intake pattern of major lipid-related nutrients increased significantly after the education period. CONCLUSIONS: These results indicate that the intake of lipid-related nutrients can be decreased by educating individual subjects about nutrition and the effects are maintained for at least 24 weeks.