Prediction of subcellular locations of proteins: where to proceed?

Since the proposal of the signal hypothesis on protein subcellular sorting, a number of computational analyses have been performed in this field. A typical example is the development of prediction algorithms for the subcellular localization sites of input protein sequences. In this review, we mainly focus on the biological grounds of the prediction methods rather than the algorithmic issues because we believe the former will be more fruitful for future development. Recent advances on the study of protein sorting signals will hopefully be incorporated into future prediction methods. Unfortunately, many of the state-of-the-art methods are published without sufficient objective tests. In fact, a simple test employed in this article shows that the performance of specifically developed predictors is not significantly better than that of a homology search. We suspect that this is a general problem associated with the interpretation of genome sequences, which have evolved through gene duplication and speciation.     

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.     

Floral distribution,clonal structure,and their effects on pollination success in a self-incompatible <Emphasis Type="Italic">Convallaria keiskei</Emphasis> population in northern Japan

In plant species, when clonal growth produces a patchy structure and flowering ramets are clustered, the amount of pollen contributing to reproductive success is often regulated by pollinator efficiency and geitonogamy. The spatial population structure may influence reproductive success. We examined the clonal structure, the spatial ramet distribution, and their combined effects on fruit set in a natural population of the insect-pollinated, self-incompatible clonal herb, Convallaria keiskei, in northern Japan. The number of shoots, flowers, and fruits in 1-m² quadrats were counted at every 5 m grid point in an established 100 × 90-m study plot. From all the quadrats where shoots existed, leaf samples were collected for allozyme analysis. Using the two spatial parameters of flowering ramet densities and genotypes, we then constructed individual-based fruit-set models. A total of 236 quadrats contained shoots, and 135 contained flowering ramets, which indicated expanded distribution of this plant throughout the study plot, while shoots, flowers and fruits all showed clustering distributions. Allozyme analysis of 282 samples revealed 94 multilocus genotypes. The largest clone extended to more than 40 m, whereas 56 genotypes were detected in only one sample. Several large clones and many small clones were distributed close to each other. Fine-scale spatial modelling revealed that the neighbouring flower numbers of different genotypes, compared with local genet or flower diversity, more influenced fruit set, in which the range of the neighbour was 14.5 m. These findings indicate that the compatible pollen dispersed by insect pollinators has a significant effect on sexual reproduction, in this C. keiskei population. Consequently, the spatial structure, which includes both genet distribution and clonal expansion by ramets, had a significant effect on pollination success.     

Structural and spatial associations between Fe, O, and C in the network structure of the Leptothrix ochracea sheath surface

The structural and spatial associations of Fe with O and C in the outer coat fibers of the Leptothrix ochracea sheath were shown to be substantially similar to the stalk fibers of Gallionella ferruginea, i.e., a central C core, probably of bacterial origin, and aquatic Fe interacting with O at the surface of the core.     

Improvement of σ1 receptor affinity by late-stage C–H-bond arylation of spirocyclic lactones

The direct C–H-bond arylation of the complex spirocyclic lactones 13, 14, and 18 allows the introduction of diverse aryl moieties in the last step of the synthesis. A selective α-arylation of the thiophene moiety was performed with the catalytic system PdCl₂/2,2′-bipyridyl/Ag₂CO₃, whereas the β-position of the thiophene ring was addressed by using the alternative catalytic system PdCl₂/P[OCH(CF₃)₂]₃/Ag₂CO₃. Due to electronic and steric reasons the arylation of the five-membered lactone 18 occurred in both α-positions providing 4′-mono-, 6′-mono- and 4′,6′-diarylated thiophenes 22–26a–c. Compounds with an additional aryl moiety at the ‘upper left (top)’ position (1′-position of 13, 3′-position of 14, 4′-position of 18) showed increased σ₁ affinity compared to the non-arylated parent compounds. A phenyl moiety at the ‘left’ position (2′-position in 20a) also increased the σ₁ affinity but to a lower extent. A considerable reduction of σ₁ affinity was observed after introducing an aryl moiety in 6′-position of 18, which might result from shielding the tertiary amine, which is crucial for interaction with the σ₁ receptor. The discussion of the experimental results is supported by high-level quantum chemical DFT-calculations of the NBO-charges of 13 and 18 and the relative energies of the related arylated products.     

On the measurement of species diversity incorporating species differences

When pairwise differences (relatedness) between species are numerically given, the average of the species differences weighted by relative frequencies can be used as a species diversity index. This paper first theoretically develops the indices of this type, then applies them to forestry data. As examples of diversity indices, this paper explores the taxonomic diversity and the newly introduced amino acid diversity, which is a modification of the nucleotide diversity in genetics. The first, mathematical part shows that both indices can be decomposed into three inner factors; evenness of relative frequencies (=the Simpson index), the simple average over species differences regardless of relative frequencies, and the taxonomic or genetic balance in relative frequencies. The taxonomic diversity has another decomposition: the sum over the Simpson indices at all the taxonomic levels. The second part examines the effects of different forest management techniques on diversity. It is shown that a thinning operation for promoting survival of specific desirable species also contributed to increasing the taxonomic diversity. If we calculated only conventional indices that do not incorporate species relatedness, we would simply conclude that the thinning did not significantly affect the diversity. The theoretical developments of the first part complement the result, leading us to a better interpretation about contrasting vegetation structures. The mathematical results also reveal that the amino acid diversity involves redundant species, which is undesirable when measuring diversity; hence, this index is used to demonstrates crucial points when we introduce species relatedness. The results suggest further possibilities of applying diversity indices incorporating species differences to a variety of ecological studies.     

Critical roles of Asp270 and Trp273 in the α-repeat of the carbohydrate-binding module of endo-1,3-β-glucanase for laminarin-binding avidity

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10⁶ M⁻¹ and 10⁴ M⁻¹, respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10⁴ M⁻¹, and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.     

Claudin-2 knockdown decreases matrix metalloproteinase-9 activity and cell migration via suppression of nuclear Sp1 in A549 cells

AimsClaudin expression is altered in lung cancer, but the pathophysiological role of claudin is not well understood. We examined the effect of claudin-2 expression on cell migration using human adenocarcinoma A549 cells.Main methodsThe mRNA level was measured by real time polymerase chain reaction. To knockdown claudin-2 expression, we made the cells expressing doxycycline-inducible claudin-2 shRNA vector. The protein level was examined by Western blotting. Cell migration was measured by wound-healing assay. The enzymatic activity of MMP-9 was assessed by gelatin zymography.Key findingsIn A549 cells, claudin-2 expression was higher than in normal lung tissue. Claudin-2 knockdown did not affect the expression of other junctional proteins including claudin-1, occludin and E-cadherin. Claudin-2 knockdown decreased cell migration concomitant with a decrease in the mRNA level and enzymatic activity of MMP-9. The expression level of Sp1 in the nuclei was decreased by claudin-2 knockdown. In contrast, the expression levels of c-Fos, c-Jun and NF-kB p65 in the nuclei were not changed by claudin-2 knockdown. The knockdown of Sp1 expression by siRNA decreased cell migration, and the mRNA expression, enzymatic activity, and promoter activity of MMP-9.SignificanceClaudin-2 may increase the mRNA level and enzymatic activity of MMP-9 mediated by the elevation of nuclear distribution of Sp1, resulting in the up-regulation of A549 cell migration.     

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

Mammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.