Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31.     

Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.     

Visualization of Sialidase Activity in Mammalian Tissues and Cancer Detection with a Novel Fluorescent Sialidase Substrate

Sialidase removes sialic acid from sialoglycoconjugates and plays crucial roles in many physiological and pathological processes. Various human cancers express an abnormally high level of the plasma membrane-associated sialidase isoform.Visualization of sialidase activity in living mammalian tissues would be useful not only for understanding sialidase functions but also for cancer diagnosis. However, since enzyme activity of mammalian sialidase is remarkably weak compared with that of bacterial and viral sialidases, it has been difficult to detect sialidase activity in mammalian tissues. We synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in acute rat brain slices. Cancer cells implanted orthotopically in mouse colons and human colon cancers (stages T3-T4) were also clearly detected with BTP-Neu5Ac. The results suggest that BTP-Neu5Ac is useful for histochemical imaging of sialidase activities.     

A Prospective Stratified Case-Cohort Study on Statins and Multiple Adverse Events in Japan

Purpose

To assess the association between statins and diverse adverse events in Japanese population.

Methods

New users of statin who started statin after 6-month period of non-use were identified in 68 hospitals between January 2008 and July 2010. In addition to the random sample subcohort, we selected additional subcohort members to make the stratified sample subcohort have at least one patient in all subgroups stratified by each combination of statin and hospital. By abstraction from medical records, detailed information was obtained for all potential cases and pre-selected subcohort members. The event review committee consisting of 3 specialists judged whether possible cases met the definition of one of the adverse events of interest, and for adjudicated cases the committee further judged whether statin was a certain, probable or possible cause of the occurrence of the event. Adjusted for covariates including age, gender, status of “switcher”, use of high daily dose and comorbidities at baseline, hazard ratio (HR) was estimated by the Cox proportional hazards model with Barlow’s weighting method. Data were also analyzed by the method proposed by Breslow in 2009.

Results

A total of 6,877 new users of a statin were identified (median age: 66 years; males: 52%). The hazard ratios of increase in serum creatinine for atorvastatin and fluvastatin have wide confidence intervals, but both of the point estimates were around 2.5. Estimates of hazard ratios by the method of Barlow (1999) were similar to those by the method of Breslow (2009).

Conclusions

Use of statin was not associated with a significant increased risk for renal, liver and muscle events. However, the hazard ratio of increase in serum creatinine tended to be high with atorvastatin and fluvastatin to require further studies.     

Structural Property of DNA That Migrates Faster in Gel Electrophoresis,as Deduced by CD Spectroscopy

Bent DNAs are known to migrate slower than ordinary DNA in non-denaturing polyacrylamide gel electrophoresis. In contrast, several satellite DNAs have been shown to migrate fast. The structural property that causes the fast migration, however, is not clarified so far on molecular basis. We have investigated the structural property of a satellite DNA, which contains consecutive purine sequences and migrates faster in gel, by CD spectroscopy. Partial formation of an A-form–like structure has been suggested. Reduction in DNA length due to the formation of the A-form–like structure may be responsible for the fast migration. The pronounced rigidity of DNA may also contribute to the behavior.     

Pattern Dynamics in Adaxial-Abaxial Specific Gene Expression Are Modulated by a Plastid Retrograde Signal during Arabidopsis thaliana Leaf Development

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.     

Compound Heterozygosity of the Functionally Null Cdh23v-ngt and Hypomorphic Cdh23ahl Alleles Leads to Early-onset Progressive Hearing Loss in Mice

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23^ahl allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23^v-ngt) null allele with mice carrying the hypomorphic Cdh23^ahl allele on the C57BL/6J background, and we then analyzed the animals’ balance and hearing phenotypes. Although the Cdh23^v-ngt/ahl compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23^ahl/ahl homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23^v-ngt/ahl mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23^v-ngt/ahl mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.     

Mating disruption of the carpenter moth,Cossus insularis (Staudinger) (Lepidoptera: Cossidae) with synthetic sex pheromone in Japanese pear orchards

Mating disruption of the carpenter moth, Cossus insularis Staudinger (Lepidoptera: Cossidae), with a synthetic version of its sex pheromone, a mixture of (E)-3-tetradecenyl acetate and (Z)-3-tetradecenyl acetate, was tested for three successive years in Japanese pear (Pyrus pyrifolia var. culta) orchards. Pheromone trap catches, percentage mating of tethered females, and tree damage were measured in both the pheromone-treated and untreated control orchards. The attraction of male adults to pheromone traps was completely disrupted, and the mating of the tethered females was completely inhibited by the treatment of synthetic pheromones. The percentage of damaged trees in the pheromone-treated orchard decreased over the course of the experiment, while the damage percentage did not decrease in the untreated orchard. These results show that mating disruption with the synthetic sex pheromone is promising for the reduction of damage caused by C. insularis in apple and Japanese pear orchards.