SR proteins induce alternative exon skipping through their activities on the flanking constitutive exons

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.     

Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts

Animals with a targeted disruption of genes can be produced by somatic cell nuclear transfer (SCNT). However, difficulties in clonal selection of somatic cells with a targeted mutation often result in heterogeneous nuclear donor cells, including gene-targeted and non-targeted cells, and impose a risk of producing undesired wildtype cloned animals after SCNT. In addition, the efficiency of cloning by SCNT has remained extremely low. Most cloned embryos die in utero, and the few that develop to term show a high incidence of postnatal death and abnormalities. In the present study, resurrection of an alpha-1,3-galactosyltransferase (αGT) gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts was attempted. Three cloned piglets were produced from the first round of SCNT, including one stillborn and two who died immediately after birth due to respiratory distress syndrome and cardiac dysfunction. Among the three piglets, two were confirmed to be αGT gene-targeted. Fibroblasts derived from postmortem ear skin biopsies were used as nuclear donor cells for the second round of SCNT, and a piglet was produced. As expected, PCR and Southern analyses confirmed that the piglet produced from recloning was αGT gene-targeted. Currently, the piglet is fourteen months of age, and no overt health problems have been observed. Results from the present study demonstrate that loss of an invaluable animal, such as a gene-targeted miniature pig, may be rescued by recloning, with assurance of the desired genetic modification.     

Effects of wearing two different types of clothing on body temperatures during and after exercise

The experiment was conducted to investigate the human thermoregulatory responses during rest, exercise and recovery atT _a 20°C and 60% R.H. under the conditions of wearing two different types of clothing. Six healthy men wore two types of clothing: one covering the whole body area except the head (Type A, weight 1656 g), and the other covering only the trunk, upper arms and thighs (Type B, weight 996 g). The level of rectal temperature was kept significantly higher in Type B than in Type A during rest and recovery. The increased and decreased rates of rectal temperature during exercise and recovery were significantly greater in Type A than in Type B, respectively. These findings are discussed from the viewpoint of the differences of skin temperatures of the extremities between Type A and Type B.     

Starch and the Control of Kernel Number in Maize at Low Water Potentials

After reproduction is initiated in plants, subsequent reproductive development is sometimes interrupted, which decreases the final number of seeds and fruits. We subjected maize (Zea mays L.) to low water potentials (psi(w)) that frequently cause this kind of failure. We observed metabolite pools and enzyme activities in the developing ovaries while we manipulated the sugar stream by feeding sucrose (Suc) to the stems. Low psi(w) imposed for 5 d around pollination allowed embryos to form, but abortion occurred and kernel number decreased markedly. The ovary contained starch that nearly disappeared during this abortion. Analyses showed that all of the intermediates in starch synthesis were depleted. However, when labeled Suc was fed to the stems, label arrived at the ovaries. Solute accumulated and caused osmotic adjustment. Suc accumulated, but other intermediates did not, showing that a partial block in starch synthesis occurred at the first step in Suc utilization. This step was mediated by invertase, which had low activity. Because of the block, Suc feeding only partially prevented starch disappearance and abortion. These results indicate that young embryos abort when the sugar stream is interrupted sufficiently to deplete starch during early ovary development, and this abortion results in a loss of mature seeds and fruits. At low psi(w), maintaining the sugar stream partially prevented the abortion, but invertase regulated the synthesis of ovary starch and partially prevented full recovery.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.     

Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.     

Genetic differences between broodstock and offspring of seven-band grouper in a hatchery

The seven-band grouper (Epinephelus septemfasciatus) is an important fishery resource of a target for prospective aquaculture diversification and maintenance of stock quality is thus important. To explore the sustainability of fry production, genetic variations in 83 seven-band groupers from two broodstock and offspring populations of a hatchery strain were analyzed using 13 polymorphic nuclear microsatellite DNA loci; 133 alleles were identified. Allelic variability ranged from 4 to 18 in the broodstock and from 3 to 11 in the offspring. The average observed and expected heterozygosities were 0.669 and 0.734 in broodstock and 0.674 and 0.649 in offspring, respectively. Although no statistically significant reductions in heterozygosity or allelic diversity were evident in offspring, considerable loss of rare alleles was apparent. The broodstock and offspring populations exhibited significant genetic differences (F _ST = 0.033, P < 0.001) indicating that genetic drift has likely promoted differentiation between the two populations, which may have negative effects on sustainable fry production. Therefore, genetic variations between broodstock and offspring should be monitored, and inbreeding should be controlled, to ensure the success of commercial breeding programs. Our data provide a useful genetic basis for future planning of sustainable culture and management of E. septemfasciatus in fisheries.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.