Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.     

Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma

The oxidative susceptibility of plasma obtained from rats after intragastric administration of quercetin was studied to know whether or not quercetin acts as an in vivo antioxidant after metabolic conversion. Quercetin was raised in the rat blood plasma essentially as glucuronide and/or sulfate conjugates. The plasma obtained from rats after quercetin administration was more resistant against copper sulfate-induced lipid peroxidation than the control plasma on the basis of the accumulation of cholesteryl ester hydroperoxides and the consumption of α-tocopherol. The results strongly suggest that some conjugated metabolites of quercetin act as effective antioxidants when plasma is subject to metal ion-induced lipid peroxidation.     

Work flow control in the flexible flow line

This research involves the development and evaluation of a part flow control model for a type of flexible manufacturing system (FMS) called a dedicated flexible flow line (FFL). In the FFL, all part types flow along the same path between successive machine groups. The specific objective of the part flow control model for the FFL is to minimize makespan for a given set of parts produced in a FFL near-term schedule, given fixed available buffer constraints. The control model developed in this research involved the repeated, real-time execution of a mathematical programming algorithm. The algorithm attempts to release the right mix of parts at the tight time to keep the FFL operating smoothly. The focus of the approach is directed toward managing WIP buffers for each machine group queue. The algorithm specifically incorporates stochastic disturbance factors such as machine failures. Through a limited number of simulation experiments, performance of the control model is shown to be superior to other parts releasing and control methods reported in the literature.     

Expression of DBC1 and Androgen Receptor Predict Poor Prognosis in Diffuse Large B Cell Lymphoma

BACKGROUND: Recently, deleted in breast cancer 1 (DBC1) has been suggested as a poor prognostic indicator of various human cancers and may possibly have a role as a coactivator of androgen receptor (AR). However, their roles in lymphoma are still unknown. MATERIALS AND METHODS: We investigated the effect of the expression of DBC1 and AR in diffuse large B cell lymphoma (DLBCL). Immunohistochemical expression of DBC1 and AR were evaluated in 101 DLBCL samples by tissue microarray. RESULTS: Positive expression of DBC1 and AR was seen in 73% and 70% of DLBCL, respectively. In total DLBCL patients, DBC1 and AR expression were significantly associated with high clinical stage, elevated serum lactate dehydrogenase levels, and high international prognostic index scores, and they predicted shorter overall survival (OS) and relapse-free survival (RFS) by univariate analysis. DBC1 expression was also an independent prognostic indicator by multivariate analysis (OS, P = .017; RFS, P = .004). Especially, both DBC1 and AR expression significantly correlated with shorter OS and RFS in non-germinal center B cell (non-GCB)-type DLBCL by univariate analysis. In multivariate analysis, DBC1 expression was an independent prognostic predictor for OS (P = .035) and AR expression significantly correlated with RFS (P = .005). CONCLUSION: We demonstrate that the expression of DBC1 and AR are significant prognostic indicators for DLBCL patients, especially for unfavorable non-GCB-type DLBCL.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013     

WDNM1 is Associated with Differentiation and Apoptosis of Mammary Epithelial Cells

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFα treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFα induced WDNM1 expression showed the NFκB-dependent mechanism since it's expression was abrogated in IκBαM (super-repressor of NFκB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFα-NFκB signal pathway, is associated with HC11 cell apoptosis.     

Development of recombinant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> producing virus-like particles of a fish nervous necrosis virus

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.     

Improved growth and acclimatization of somatic embryo-derived Oplopanax elatus plantlets by ventilated photoautotrophic culture

To improve large-scale in vitro production of Oplopanax elatus Nakai, we cultured somatic embryo-derived plantlets under a heterotrophic condition (semi-solid culture with sucrose), photoautotrophic condition (semi-solid culture without sucrose), or modified photoautotrophic condition (liquid culture with forced ventilation). The plantlets grown under the modified photoautotrophic condition had more leaves as well as higher chlorophyll content, and higher net photosynthetic rate than those grown under the conventional conditions. Further, the photoautotrophically grown plantlets acclimatized better and sooner upon ex vitro transplantation than did the conventionally cultured plantlets. Consequently, a photoautotrophic culture method with forced ventilation is effective for enhancing the growth and acclimatization of O. elatus.     

Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line

Spinal muscular atrophy is a genetic disease in which the SMN1 gene is deleted. The SMN2 gene exists in all of the patients. Alternative splicing of these two genes are different. More than 90% of exon 7 included form is produced from SMN1 pre-mRNA, whereas only ～20% of exon 7 included form is produced from SMN2 pre-mRNA. Only exon 7 inclusion form produces functional protein. Exon 7 skipped SMN isoform is unstable. Here we constructed a GFP reporter system that recapitulates the alternative splicing of SMN1 and SMN2 pre-mRNA. We designed a system in which GFP protein is expressed only when exon 7 of is included in alternative splicing. The stable cell that expresses SMN1-GFP produces ～4 times more GFP protein than the stable cell line that expresses SMN2-GFP; as demonstrated by microscopy, FACS analysis and immunoblotting. In addition the ratio of exon 7 inclusion and skipping of SMN1-GFP and SMN2-GFP pre-mRNA was similar to endogenous SMN1 and SMN2 pre-mRNA as shown in RT-PCR. Furthermore the knockdown with hnRNP A1 shRNA, a known protein which promotes exon 7 skipping of SMN2, induces exon 7 inclusion of exon 7 in SMN2-GFP pre-mRNA in SMN2-GFP cell line. We conclude that we have established the stable cell lines that recapitulate alternative splicing of the SMN1 and SMN2 genes. The stable cell line can be used to identify the trans-acting elements with siRNA.