Genomic adaptation of flowering‐time genes during the expansion of rice cultivation area

The diversification of flowering time in response to natural environments is critical for the spread of crops to diverse geographic regions. In contrast with recent advances in understanding the molecular basis of photoperiodic flowering in rice (Oryza sativa), little is known about how flowering‐time diversification is structured within rice subspecies. By analyzing genome sequencing data and a set of 429 chromosome segment substitution lines (CSSLs) originating from 10 diverse rice accessions with wide distributions, we revealed diverse effects of allelic variations for common flowering‐time quantitative trait loci in the recipient's background. Although functional variations associated with a few loci corresponded to standing variations among subspecies, the identified functional nucleotide polymorphisms occurred recently after rice subgroup differentiation, indicating that the functional diversity of flowering‐time gene sequences was not particularly associated with phylogenetic relationship between rice subspecies. Intensive analysis of the Hd1 genomic region identified the signature of an early introgression of the Hd1 with key mutation(s) in aus and temperate japonica accessions. Our data suggested that, after such key introgressions, new mutations were selected and accelerated the flowering‐time diversity within subspecies during the expansion of rice cultivation area. This finding may imply that new genome‐wide changes for flowering‐time adaptation are one of the critical determinants for establishing genomic architecture of local rice subgroups. In‐depth analyses of various rice genomes coupling with the genetically confirmed phenotypic changes in a large set of CSSLs enabled us to demonstrate how rice genome dynamics has coordinated with the adaptation of cultivated rice during the expansion of cultivation area.     

DNA methylation analysis from saliva samples for epidemiological studies

Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.     

Crosstalk between Na+,K+-ATPase and a volume-regulated anion channel in membrane microdomains of human cancer cells

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na⁺,K⁺-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na⁺,K⁺-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na⁺,K⁺-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na⁺,K⁺-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.     

Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies

Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that framework region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized antibody. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool.     

Multiple introgression events and range shifts in Schizocodon (Diapensiaceae) during the Pleistocene

Range shifts during the Pleistocene shaped the unique phylogeographical structures of numerous species. Accompanying species migration, sister taxa may have experienced multiple introgression events. Here, we report the signature of introgression events in multiple areas in Schizocodon, herbs endemic to Japan, using amplified fragment length polymorphism (AFLP) fingerprinting and plastid DNA haplotyping in 48 populations. Although the present distributions of S. soldanelloides and S. ilicifolius are mainly allopatric, the species share plastid DNA haplotypes in each region (north‐eastern, north‐central, south‐central and south‐western Japan); in contrast, the specific groups were highly supported by AFLP analyses. These results support the occurrence of multiple introgression events in Schizocodon. Notably, the disjunct plastid haplotypes found only in S. ilicifolius var. intercedens suggest complete plastid DNA replacement at local areas from S. soldanelloides into S. ilicifolius var. ilicifolius. Furthermore, we found that S. soldanelloides experienced range contraction and expansion during glacial and interglacial cycles based on mismatch distribution analysis and ecological niche modelling. Based on several pieces of evidence, our study supports the idea that historical range shifts associated with Pleistocene climatic oscillations favoured multiple and regional introgression events in Schizocodon. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 46–63.     

Identification of CtpL as a Chromosomally Encoded Chemoreceptor for 4-Chloroaniline and Catechol in Pseudomonas aeruginosa PAO1

Bacterial chemotaxis influences the ability of bacteria to survive and thrive in most environments, including polluted ones. Despite numerous reports of the phenotypic characterization of chemotactic bacteria, only a few molecular details of chemoreceptors for aromatic pollutants have been described. In this study, the molecular basis of chemotaxis toward an environmentally toxic chlorinated aromatic pollutant, 4-chloroaniline (4CA), was evaluated. Among the three Pseudomonas spp. tested, Pseudomonas aeruginosa PAO1 exhibited positive chemotaxis both to the nonmetabolizable 4CA, where 4-chloroacetanilide was formed as a dead-end transformation product, and to the metabolizable catechol. Molecular analysis of all 26 mutants with a disrupted methyl-accepting chemotaxis gene revealed that CtpL, a chromosomally encoded chemoreceptor, was responsible for the positive chemotactic response toward 4CA. Since CtpL has previously been described to be a major chemoreceptor for inorganic phosphate at low concentrations in PAO1, this report describes a fortuitous ability of CtpL to function toward aromatic pollutants. In addition, its regulation not only was dependent on the presence of the chemoattractant inducer but also was regulated by conditions of phosphate starvation. These results expand the range of known chemotactic transducers and their function in the environmental bacterium PAO1.     

Neurons innervating the lamina in the butterfly, Papilio xuthus

The butterfly Papilio xuthus has compound eyes with three types of ommatidia. Each type houses nine spectrally heterogeneous photoreceptors (R1–R9) that are divided into six spectral classes: ultraviolet, violet, blue, green, red, and broad-band. Analysis of color discrimination has shown that P. xuthus uses the ultraviolet, blue, green, and red receptors for foraging. The ultraviolet and blue receptors are long visual fibers terminating in the medulla, whereas the green and red receptors are short visual fibers terminating in the lamina. This suggests that processing of wavelength information begins in the lamina in P. xuthus, unlike in flies. To establish the anatomical basis of color discrimination mechanisms, we examined neurons innervating the lamina by injecting Neurobiotin into this neuropil. We found that in addition to photoreceptors and lamina monopolar cells, three distinct groups of cells project fibers into the lamina. Their cell bodies are located (1) at the anterior rim of the medulla, (2) between the proximal surface of the medulla and lobula plate, and (3) in the medulla cell body rind. Neurobiotin injection also labeled distinct terminals in medulla layers 1, 2, 3, 4 and 5. Terminals in layer 4 belong to the long visual fibers (R1, 2 and 9), while arbors in layers 1, 2 and 3 probably correspond to terminals of three subtypes of lamina monopolar cells, respectively. Immunocytochemistry coupled with Neurobiotin injection revealed their transmitter candidates; neurons in (1) and a subset of neurons in (2) are immunoreactive to anti-serotonin and anti-γ-aminobutyric acid, respectively.     

Serum heat shock protein 47 levels in patients with drug-induced lung disease

Background

Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. We recently reported that HSP47 serum levels were markedly higher in patients with acute exacerbations of idiopathic pulmonary fibrosis (IPF) when compared with patients with stable IPF, suggesting that serum HSP47 levels correlate with interstitial pneumonia activity. The aim of this study was to evaluate serum HSP47 levels in patients with drug-induced lung disease (DILD).

Methods

Findings from high-resolution computed tomographic chest scans of 47 patients with DILD were classified into one of four predominant patterns: organizing pneumonia (OP) (n = 4), nonspecific interstitial pneumonia (NSIP) (n = 24), hypersensitivity pneumonitis (HP) (n = 11), and diffuse alveolar damage (DAD) (n = 8). Serum levels of HSP47, Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-A, and SP-D were measured in these patients.

Results

The PaO₂/fraction of inspired oxygen (FiO₂) (P/F) ratios were significantly lower and the alveolar-arterial difference of oxygen (A-a DO₂) was significantly higher in the DAD group than in the other groups. Patients with DAD had the worst outcomes among the different subgroups. Patients in the DAD group had significantly higher serum HSP47 levels than those in other groups. Receiver operating characteristic curves revealed that HSP47 was superior to KL-6, SP-A, and SP-D for discriminating between the DAD group and the other groups. The cut-off level for HSP47 that resulted in the highest diagnostic accuracy was 1711.5 pg/mL. The sensitivity, specificity, and diagnostic accuracy were 87.5%, 97.4%, and 95.7%, respectively. Serum levels of HSP47 in the group of patients requiring glucocorticoids were significantly higher than those in patients who experienced clinical improvement without glucocorticoid administration. Serum HSP47 levels also significantly correlated with various respiratory parameters.

Conclusion

This study demonstrated that serum HSP47 levels were elevated in patients with DILD with a DAD pattern who had the worst outcomes among the different subgroups, and that this was correlated with P/F ratio and A-a DO₂.     

On the Role of Disulfide Bonds in Polymerization of Soybean 7S Globulin during Storage

Modification of soybean 7S globulin with N-ethylmaleimide (NEM) was effective for preventing humidity-induced insolubilization during storage. The sulfhydryl-disulfide interchange reaction and/or oxidation of the sulfhydryl groups to form disulfide bonds closely related to the polymerization of the 7S globulin. A dimer, consisting of α′ and α subunits linked with disulfide bonds, was observed in the polymerized 7S globulin fractions, but this dimer was not readily apparent in the NEM-7S globulin. The formation of the dimer certainly initiates the insolubilization of the 7S globulin during storage. Although the β subunit had sulfhydryl groups, it did not take a part in the formation of the dimer.