Thermal stability of oligodeoxynucleotide duplexes containing l-deoxynucleotide at termini

The effects of substituting l-deoxynucleotide for d-deoxynucleotide at duplex termini were evaluated and the terminal substitutions were found to show much less effects on duplex destabilization and to show a similar tendency in base pairing selectivity, compared with internal chiral substitutions.     

Structural basis for regulation of poly‐SUMO chain by a SUMO‐like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Cruciform extrusion propensity of human translocation-mediating palindromic AT-rich repeats

There is an emerging consensus that secondary structures of DNA have the potential for genomic instability. Palindromic AT-rich repeats (PATRRs) are a characteristic sequence identified at each breakpoint of the recurrent constitutional t(11;22) and t(17;22) translocations in humans, named PATRR22 (~600bp), PATRR11 (~450bp) and PATRR17 (~190bp). The secondary structure-forming propensity in vitro and the instability in vivo have been experimentally evaluated for various PATRRs that differ regarding their size and symmetry. At physiological ionic strength, a cruciform structure is most frequently observed for the symmetric PATRR22, less often for the symmetric PATRR11, but not for the other PATRRs. In wild-type E. coli, only these two PATRRs undergo extensive instability, consistent with the relatively high incidence of the t(11;22) in humans. The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo. Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo. Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.     

Akt is a neutral amplifier for Th cell differentiation

Both CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4(+) T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4(+) T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4(+) T cells. E40K also facilitated Th1 differentiation in CD4(+) T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation.     

Palindrome-mediated chromosomal translocations in humans

Recently, it has emerged that palindrome-mediated genomic instability contributes to a diverse group of genomic rearrangements including translocations, deletions, and amplifications. One of the best studied examples is the recurrent t(11;22) constitutional translocation in humans that has been well documented to be mediated by palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11. De novo examples of the translocation are detected at a high frequency in sperm samples from normal healthy males, but not in lymphoblasts or fibroblasts. Cloned breakpoint sequences preferentially form a cruciform configuration in vitro. Analysis of the junction fragments implicates frequent double-strand-breaks (DSBs) at the center of both palindromic regions, followed by repair through the non-homologous end joining (NHEJ) pathway. We propose that the PATRR adopts a cruciform structure in male meiotic cells, creating genomic instability that leads to the recurrent translocation.     

Optimal vibration stimulation to the neck extensor muscles using hydraulic vibrators to shorten saccadic reaction time

Optimal vibration stimulation to the neck extensor muscles using hydraulic vibrators to shorten the saccadic reaction time was examined. Subjects were 14 healthy young adults. Visual targets (LEDs) were located 10 degrees left and right of a central point. The targets were alternately lit for random durations of 2-4 seconds in a resting neck condition and various vibration conditions, and saccadic reaction times were measured. Vibration amplitude was 0.5 mm in every condition. The upper trapezius muscles were vibrated at 40, 60, 80, and 100 Hz in a sub-maximum stretch condition in which the muscles were stretched at 70% of maximum stretch. In addition, the muscles were vibrated at 60 Hz with the muscles maximally stretched, with 70% vertical pressure without stretching, and with vibration applied to the skin in the same area as the muscle vibration. At 60, 80, and 100 Hz at 70% maximum stretch, saccadic reaction time shortened significantly compared with the resting neck condition. However, no significant difference in the reaction time was observed among the frequencies. The saccadic reaction times in the maximum stretch condition, muscle pressure condition, and skin contact condition did not differ significantly from that in the resting neck condition. Vibration stimulation to the trapezius with 60-100 Hz frequencies at 0.5 mm amplitude in the sub-maximum stretch condition was effective for shortening saccadic reaction time. The main mechanism appears to be Ia information originating from the muscle spindle.     

Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo

Alternative splicing of pre-mRNAs allows multicellular organisms to create a huge diversity of proteomes from a finite number of genes. But extensive studies in vitro or in cultured cells have not fully explained the regulation mechanisms of tissue-specific or developmentally regulated alternative splicing in living organisms. Here we report a transgenic reporter system that allows visualization of expression profiles of mutually exclusive exons in Caenorhabditis elegans. Reporters for egl-15 exons 5A and 5B showed tissue-specific profiles, and we isolated mutants defective in the tissue specificity. We identified alternative-splicing defective-1 (asd-1), encoding a new RNA-binding protein of the evolutionarily conserved Fox-1 family, as a regulator of the egl-15 reporter. Furthermore, an asd-1;fox-1 double mutant was defective in the expression of endogenous egl-15 (5A) and phenocopied egl-15 (5A) mutant. This transgenic reporter system can be a powerful experimental tool for the comprehensive study of expression profiles and regulation mechanisms of alternative splicing in metazoans.     

Crystal structure of SUMO-3-modified thymine-DNA glycosylase

Modification of cellular proteins by the small ubiquitin-like modifier SUMO is important in regulating various cellular events. Many different nuclear proteins are targeted by SUMO, and the functional consequences of this modification are diverse. For most proteins, however, the functional and structural consequences of modification by specific SUMO isomers are unclear. Conjugation of SUMO to thymine-DNA glycosylase (TDG) induces the dissociation of TDG from its product DNA. Structure determination of the TDG central region conjugated to SUMO-1 previously suggested a mechanism in which the SUMOylation-induced conformational change in the C-terminal region of TDG releases TDG from tight binding to its product DNA. Here, we have determined the crystal structure of the central region of TDG conjugated to SUMO-3. The overall structure of SUMO-3-conjugated TDG is similar to the previously reported structure of TDG conjugated to SUMO-1, despite the relatively low level of amino acid sequence similarity between SUMO-3 and SUMO-1. The two structures revealed that the sequence of TDG that resembles the SUMO-binding motif (SBM) can form an intermolecular beta-sheet with either SUMO-1 or SUMO-3. Structural comparison with the canonical SBM shows that this SBM-like sequence of TDG retains all of the characteristic interactions of the SBM, indicating sequence diversity in the SBM.     

High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81

The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8+/-1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins.