Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

© 2014 Fujikawa et al. Background: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome

Connective Tissue Growth Factor Gene Expression in Tissue Sections From Localized Scleroderma, Keloid, and Other Fibrotic Skin Disorders

Connective tissue growth factor (CTGF) is a novel peptide that exhibits platelet-derived growth factor-like activities and is produced by skin fibroblasts after activation with transforming growth factor-β. Coordinate expression of transforming growth factor-β followed by CTGF during wound repair suggests a cascade process for control of tissue regeneration. We recently reported a significant correlation between CTGF mRNA expression and histologic sclerosis in systemic sclerosis. To confirm the relation between CTGF and skin fibrosis, we investigated CTGF gene expression in tissue sections from patients with localized scleroderma, keloid, and other sclerotic skin disorders using nonradioactive in situ hybridization. In localized scleroderma, the fibroblasts with positive signals for CTGF mRNA were scattered throughout the sclerotic lesions with no preferential distribution around the inflammatory cells or perivascular regions, whereas the adjacent nonaffected dermis was negative for CTGF mRNA. In keloid tissue, the fibroblasts positive for CTGF mRNA were diffusely distributed, especially in the peripheral expanding lesions. In scar tissue, however, the fibroblasts in the fibrotic lesions showed partially positive signals for CTGF mRNA. In eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture, CTGF mRNA was also expressed partially in the fibroblasts of the fibrotic lesions. Our findings reinforce a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-β plays an important role in the pathogenesis of fibrosis, as it is the only inducer for CTGF identified to date

Oral health status: relationship to nutrient and food intake among 80‐year‐old Japanese adults

Objectives The aim of this cross‐sectional study was to investigate the relationship of oral health status defined on the basis of presence of posterior occluding pairs ( POP s) and adequacy of removable denture fit as determined by self‐report to nutrient and food intake among older Japanese. Methods The subjects were 353 Japanese aged 80 years in 2008 and were classified into four groups according to the number of POP s, defined as pairs of occluding natural, restored, or fixed prosthetic postcanine teeth (range: 0–8) and removable denture status. The groups were: (i) good dentition ( n  =   56; 8 POP s and no removable prosthesis), (ii) well‐fitting dentures ( n  =   158; <8 POP s with self‐reported good‐fitting dentures), (iii) ill‐fitting dentures ( n  =   70; <8 POP s with self‐reported ill‐fitting dentures), and (iv) compromised dentition ( n  =   69; <8 POP s and no removable prosthesis). Multivariable analysis of the differences in nutrient and food intake outcome variables which were collected via validated food frequency questionnaire among the four oral health status groups was conducted using general linear models. Results Intake of multiple nutrients was significantly ( P  <   0.05) lower in the group with ill‐fitting dentures or compromised dentition than in the good dentition group. Vegetable, fish, and shellfish consumption was significantly lower in the ill‐fitting dentures or compromised dentition groups. No significant differences were seen in dietary intake between the well‐fitting dentures and good dentition groups. Conclusions Dietary intake was poorer in those with self‐perceived ill‐fitting dentures or fewer POP s than among those having all POP s. Regular dental care to maintain intact dentition, as well as dental treatment to replace missing teeth and ensure adequate denture fit and function, may be important to the diet intake and subsequent nutritional status of older Japanese.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108691/1/cdoe12100.pd

Western North Pacific Integrated Physical-Biogeochemical Ocean Observation Experiment (INBOX): Part 1.Specifications and chronology of the S1-INBOX floats

An interdisciplinary project called the Western North Pacific Integrated Physical-Biogeochemical Ocean Observation Experiment (INBOX) has been conducted since 2011. In the oligotrophic subtropics south of the Kuroshio Extension near biogeochemical mooring S1 (30° N, 145° E), 18 floats, each with a dissolved oxygen sensor, have been deployed in a 150 × 150 km square area. With the horizontal (30 km) and temporal (2 days) resolution of the data, we observed an upper ocean structure associated with mesoscale eddies and ocean responses to atmospheric forcing. The data set obtained from the S1-INBOX study was used to elucidate the impacts of physical processes on biogeochemical phenomena. This article is the first in a series of articles: specific information about the floats and a chronology of events are provided

Glycine cleavage system in neurogenic regions

The glycine cleavage system (GCS) is the essential enzyme complex for degrading glycine and supplying 5,10-methylenetetrahydrofolate for DNA synthesis. Inherited deficiency of this system causes non-ketotic hyperglycinemia, characterized by severe neurological symptoms and frequent association of brain malformations. Although high levels of glycine have been considered to cause the above-mentioned problems, the detailed pathogenesis of this disease is still unknown. Here we show that GCS is abundantly expressed in rat embryonic neural stem/progenitor cells in the neuroepithelium, and this expression is transmitted to the radial glia-astrocyte lineage, with prominence in postnatal neurogenic regions. These data indicate that GCS plays important roles in neurogenesis, and suggest that disturbance of neurogenesis induced by deficiency of GCS may be the main pathogenesis of non-ketotic hyperglycinemi

Cellular Polyamine Catalogues of the Five Classes of the PhylumProteobacteria: Distributions of Homospermidine within the ClassAlphaproteobacteria, Hydroxyputrescine within the ClassBetaproteobacteria, Norspermidine within the ClassGammaproteobacteria, and Spermine within the ClassesDeltaproteobacteria and Epsilonproteobacteria

Cellular polyamines extracted from reclassified or newly validated 47 alphaproteobacteria, 46 betaproteobacteria, 96 gammaproteobacteria, 12 deltaproteobacteria and 10 epsilonproteobacteria were analyzed by high-performance liquid chromatography. Homospermidine was widely distributed within the class Alphaproteobacteria, however, homospermidine-dominant type, spermidine-dominant type and homospermidine/spermidinedominant type were found and the three triamine profiles were genus-specific. The all genera belonging to the class Betaproteobacteria, ubiquitously contained putrescine and 2- hydroxyputrescine. Triamines were absent in almost betaproteobacteria. Many genera, including psychrophilic species, of the class Gammaproteobacteria, contained putrescine and spermidine as the major polyaminenes. Diaminopropane and norspermidine were selectively distributed in several genera of the class Gammaproteobacteria. Spermidine was the major polyamine in the classes Deltaproteobacteria and Epsilonproteobacteria. Spermine was found in some thermophiles within Betaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria, suggesting that the occurrence of spermine correlate to their thermophily. Additional these polyamine catalogues serve for the classification of the phylum Proteobacteria, as a chemotaxonomic marker

Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections

We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR

Salivary Effects of Facial Vibrotactile Stimulation in Patients with Sjogren’s Syndrome and Poor Salivation

We examined the effect of vibrotactile apparatus in patients with Sjögren’s syndrome and others with reduced salivation in comparison to normal subjects. The most effective salivation in normal subjects was produced by 89 Hz vibrotactile stimulation with 9.8 μm amplitude on the parotid or submandibular glands vibrotactile stimuli. First, we examined by measuring the weight of dental cotton rolls positioned at the opening of the secretory duct for total salivation 3 min during resting, and then after 5-min intervals, the weights were measured every 3 min of vibrotactile stimulation on salivary glands. Furthermore, we measured facial temperature around vibrators after 2 min of vibration. We investigated 10 poor salivation patients with Sjögren’s syndrome (8 patients) defined by examinations (contrast study or scintigraphic test) and others (2 patients). About 50% of patients with poor salivation gained recognition for good results, although they had periods of short-term (3 months) and long-term effects (6–7 years) during recuperation. Furthermore, facial skin temperatures on both sides of parotid glands were decreased in Sjogren’s syndrome after vibration, although their temperatures were increased following recovery. Although the mechanism is not clear, we think that vibrotactile stimulation gives activation to salivary glands under the rising facial temperature