Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.     

Substrate competition and specificity at the active site of amylopullulanase from Clostridium thermohydrosulfuricum

A highly thermostable pullulanase purified from Clostridium thermohydrosulfuricum strain 39E displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved alpha-1,6 bonds in pullulan, while it showed alpha-1,4 activity against malto-oligosaccharides. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as the two competing substrates were used to distinguish the dual specificity of the enzyme from the single substrate specificity known for pullulanases and alpha-amylases.     

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification, properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^–1 sec^–1 for LA-1 and 0.8 × 10⁹ M^–1 sec^–1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^–1 sec^–1 for LA-1 and 1.2×10¹¹M^–1 sec^–1 for LA-2. Analysis of the circular dichroic spectra yields 40%-helix and 60%-turn for La-1 and 45%-helix and 55%-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

Purification and characterization of an extracellular β-xylosidase from a newly isolated Fusarium verticillioides

An extracellular β-xylosidase from a newly isolated Fusarium verticillioides (NRRL 26518) was purified to homogeneity from the culture supernatant by concentration by ultrafiltration using a 10,000 cut-off membrane, ammonium sulfate precipitation, DEAE Bio-Gel A agarose column chromatography and SP-Sephadex C-50 column chromatography. The purified β-xylosidase (specific activity, 57 U/mg protein) had a molecular weight (mol. wt.) of 94,500 and an isoelectric point at pH 7.8. The optimum temperature and pH for action of the enzyme were 65°C and 4.5, respectively. It hydrolyzes xylobiose and higher xylooligosaccharides but is inactive against xylan. The purified β-xylosidase had a K _m value of 0.85 mM (p-nitrophenol-β-D-xyloside, pH 4.5, 50°C) and was competitively inhibited by xylose with a K _i value of 6 mM. It did not require any metal ion for activity and stability. Journal of Industrial Microbiology & Biotechnology (2001) 27, 241–245. Received 20 May 2001/ Accepted in revised form 06 July 2001     

Characterization of zinc oxide nanocrystals with different morphology for application in ultraviolet‐light photocatalytic performances on rhodamine B

ZnO nanostructures of different morphology (nanorods, nano‐leaf, nanotubes) were favourably grown using a chemical precipitation process. The prepared ZnO nanostructures were characterized systematically using absorption spectroscopy, emission spectroscopy, X‐ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared studies. XRD results showed the hexagonal wurtzite phase of the synthesized ZnO nanostructures. Structural properties such as average crystallite size, lattice constants, volume of the unit cell, atomic fraction, and structural bonds were also studied. The optical band gap of the synthesized ZnO nanocrystals varied from 3.52 eV to 3.69 eV with high quantum yield of the blue emission (~420 nm). Urbach energy for ZnO nanocrystals was calculated to be 0.702 eV, 0.901 eV, and 0.993 eV for nanorods, nano‐leaf, and tube like ZnO crystals, respectively. Morphology of the fabricated nanostructures was investigated using SEM. Photocatalytic degradation of rhodamine B (Rh B) in solution under UV irradiation was explored with different ZnO morphology. Photocatalytic experiments showed that ZnO nano‐leaf had a higher degradation rate of photocatalytic activity of photodegrading Rh B compared with the other tube shape and rods shape nanostructures. The Rh B dye degraded considerably by ～79.05%, 74.41%, and 69.8% within 120 min in the presence of the as‐fabricated fern nano‐leaf, nanotubes, and nanorods of the ZnO nanocrystals at room temperature.     

The RBP-Jκ Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

Kaposi''s sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman''s disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA_1st) and all three RBP-Jκ binding sites (RTA_all) within the RTA promoter. Our results showed that RTA_1st and RTA_all recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA_1st and RTA_all viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA_1st and RTA_all recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA_1st and RTA_all recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA_1st and RTA_all recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA_1st and RTA_all recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases.