Structure and Novel Functional Mechanism of Drosophila SNF in Sex-Lethal Splicing

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar to Sex-lethal protein (SXL). The polyuridine RNA binding enables SNF directly inhibit Sxl exon 3 splicing, as the dominant negative mutant SNF¹⁶²¹ binds U1 snRNA but not polyuridine RNA. Unlike U1A, both RNA recognition motifs (RRMs) of SNF can recognize polyuridine RNA tracts independently, even though SNF and U1A share very high sequence identity and overall structure similarity. As SNF RRM1 tends to self-associate on the opposite side of the RNA binding surface, it is possible for SNF to bridge the formation of super-complexes between two introns flanking Sxl exon 3 or between a intron and U1 snRNP, which serves the molecular basis for SNF to directly regulate Sxl splicing. Taken together, a new functional model for SNF in Drosophila sex determination is proposed. The key of the new model is that SXL and SNF function similarly in promoting Sxl male-specific exon skipping with SNF being an auxiliary or backup to SXL, and it is the combined dose of SXL and SNF governs Drosophila sex determination.     

Biosynthesis of salicylic acid in plants

Salicylic acid (SA) is an important signal molecule in plants. Two pathways of SA biosynthesis have been proposed in plants. Biochemical studies using isotope feeding have suggested that plants synthesize SA from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL). Silencing of PAL genes in tobacco or chemical inhibition of PAL activity in Arabidopsis, cucumber and potato reduces pathogen-induced SA accumulation. Genetic studies, on the other hand, indicate that the bulk of SA is produced from isochorismate. In bacteria, SA is synthesized from chorismate through two reactions catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Arabidopsis contains two ICS genes but has no gene encoding proteins similar to the bacterial IPL. Thus, how SA is synthesized in plants is not fully elucidated. Two recently identified Arabidopsis genes, PBS3 and EPS1, are important for pathogen-induced SA accumulation. PBS3 encodes a member of the acyl-adenylate/thioester-forming enzyme family and EPS1 encodes a member of the BAHD acyltransferase superfamily. PBS3 and EPS1 may be directly involved in the synthesis of an important precursor or regulatory molecule for SA biosynthesis. The pathways and regulation of SA biosynthesis in plants may be more complicated than previously thought.Key words: salicylic acid biosynthesis, isochorismate synthase, phenylalanine ammonia lyase     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

The significance of CD14+ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis

Background aimsCirculating monocytes have been exploited as an important progenitor cell resource for hepatocytes in vitro and are instrumental in the removal of fibrosis. We investigated the significance of monocytes in peripheral blood stem cells (PBSC) for the treatment of liver cirrhosis.MethodsRat CD14⁺ monocytes in PBSC were mobilized with granulocyte-colony-stimulating factor (G-CSF) and harvested by magnetic cell sorting (MACS). Female rats with carbon tetrachloride (CCl₄)-induced liver cirrhosis were injected CM-DiI-labeled monocytes, CD14^? cells (1 × 10⁷ cells/rat) or saline via the portal vein.ResultsRat CD14⁺ and CD11b⁺ monocytes in PBSC were partly positive for CD34, CD45, CD44, Oct3/4 and Sox2, suggesting monocytes with progenitor capacity. Compared with CD14^? cell-infused and saline-injected rats, rats undergoing monocyte transplantation showed a gradually increased serum albumin level and decreased portal vein pressure, resulting in a significantly improved survival rate. Meanwhile, monocyte transplantation apparently attenuated liver fibrosis by analysis for fibronectin, α2-(1)-procollagen, α-smooth muscle aorta (SMA) and transforming growth factor (TGF)-β. Transplanted monocytes mainly clustered in periportal areas of liver, in which 1.8% cells expressed hepatocyte marker albumin and CK18. The expression level of hepatocyte growth factor (HGF), TGF-α, extracellular matrix (EGF) and vascular endothelial growth factor (VEGF) increased, while monocyte transplantation enhanced hepatocyte proliferation. On the other hand, the activities and expression of matrix metalloproteinases (MMP) increased while tissue inhibitor of metalloproteinase (TIMP)-1 expression significantly reduced in monocyte-transplanted livers. Some transplanted monocytes expressed MMP-9 and -13.ConclusionsThe data suggest that CD14⁺ monocytes in PBSC contribute to hepatocyte regeneration and extracellular matrix (ECM) remodeling in rat liver cirrhosis much more than CD14^? cells, and might offer a therapeutic alternative for patients with liver cirrhosis.     

A first genome assembly of the barley fungal pathogen <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis>

Background  

Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the cause of one of barley's most important diseases, net form of net blotch. Here we report the first genome assembly for this species based solely on short Solexa sequencing reads of isolate 0-1. The assembly was validated by comparison to BAC sequences, ESTs, orthologous genes and by PCR, and complemented by cytogenetic karyotyping and the first genome-wide genetic map for P. teres f. teres.     

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

Background  

Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.     

Characterization and adsorption properties of eggshells and eggshell membrane

The objective of this work was to study the chemical and physical characterization of eggshell and eggshell membrane particles prepared from the hen eggshell waste. Under the characterization measurements investigated, it was found that the pore structures of the two biomaterials belong to a typical Type II, indicating that they should be basically characteristic of nonporous materials or materials with macropores or open voids. Further, the chemical composition of the resulting eggshell particle was strongly associated with the presence of carbonate minerals from the Fourier transform infrared (FTIR) spectra. In contrast to the resulting eggshell membrane particle, the presence of functional groups of amines and amides was observable because of its chemical composition of fibrous proteins. From the isotherm data of methylene blue at 25 degrees C, the Freundlich model yielded a somewhat better fit than the Langmuir model. The adsorption isotherms revealed the eggshell biosorbents could only uptake the basic dye of less than 1.0mg/g in aqueous medium, which was attributed to their poor pore properties.     

Synthesis of (2E)-3-{2-[(substituted benzyl)oxy]phenyl}acrylaldehydes as novel anti-inflammatory agents

As part of our continuing effort for development of novel anti-inflammatory agents, the highly potential agent CCY1a, which we reported recently, was selected as lead compound to synthesize a series of its derivatives for evaluation. Most of the newly synthesized compounds exhibited superior inhibitory activity than both the lead compound and the positive control (trifluoperazine) toward fMLP-stimulated neutrophil superoxide formation. (2E)-3-[2-(Benzyloxy)-5-methoxyphenyl]-acrylaldehyde (31) was among the most potent with action mechanism different from CCY1a in that it does not act as cAMP-elevating agent but inhibits the increase in cellular Ca(2+) with greater potency.