Genome-Wide Expression Analysis in Fibroblast Cell Lines from Probands with Pallister Killian Syndrome

Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.     

Urocystis tranzscheliana,a newly recorded smut fungus onPrimula sieboldii from Japan

A smut fungus onPrimula sieboldii was newly found in Japan and identified asUrocystis tranzscheliana by comparative morphology. This species causes systemic infection ofP. sieboldii and produces sori in its ovaries.Contribution No. 121, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.     

A distinct role of the queen in coordinated workload and soil distribution in eusocial naked mole-rats

We investigated how group members achieve collective decision-making, by considering individual intrinsic behavioural rules and behavioural mechanisms for maintaining social integration. Using a simulated burrow environment, we investigated the behavioural rules of coordinated workload for soil distribution in a eusocial mammal, the naked mole-rat (Heterocephalus glaber). We tested two predictions regarding a distinct role of the queen, a socially dominant individual in the caste system: the presence of a queen would increase the workload of other caste individuals, and the cues by a queen would affect the soil distribution. In experiment 1, we placed four individuals of various castes from the same colony into an experimental burrow. Workers exhibited the highest frequency of workload compared to other castes. The presence of a queen activated the workload by other individuals. Individuals showed a consistent workload in a particular direction so as to bias the soil distribution. These results suggest that individuals have a consensus on soil distribution and that the queen plays a distinct role. In experiment 2, we placed the odour of a queen in one of four cells and observed its effect on other individuals' workload and soil distribution. Relative to other cells, individuals frequently dug in the queen cell so the amount of soil in the queen cell decreased. These results suggest that queen odour is an important cue in coordinated workload and soil distribution in this species.     

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells

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A Similar Expression Pattern of Adhesion Molecules between Intermediate TCR Cells in the Liver and Intraepithelial Lymphocytes in the Intestine

Two major populations of extrathymically differentiated T cells exist in the liver and intestine. Such T cells in the liver have TCR of intermediate intensity (i.e., intermediate TCR cells) and constitutively express IL-2 receptor β-chain (IL-2Rβ), whereas those in the intestine, especially intraepithelial lymphocytes, have TCR of bright intensity, consisting of a mixture of IL-2Rβ⁺ and IL-2Rβ^–. All mature thymocytes and thymus-derived T cells seen in the peripheral immune organs are TCR-bright⁺IL-2Rβ^– under resting conditions. When the expression pattern of adhesion molecules, including CD44, L-selectin, LFA-1 and ICAM-1, was compared among these T-cell populations, they displayed quite unique patterns of expression. All extrathymic T cells in the liver, intestine, and even other organs were CD44⁺L-selectin^– LFA-1⁺⁺ICAM-1⁺, whereas thymocytes and thymus-derived T cells were CD44^– L-selectin⁺LFA-1⁺ICAM-1^–. This inverted expression of adhesion molecules between extrathymic T cells and thymus-derived T cells might be associated with their unique tissue-localization.     

Photoreaction of tyrosin-iodinated bacteriorhodopsin at low temperature

To elucidate the role of tyrosine residues in the shift of max and the light-driven proton pump of bacteriorhodopsin~ the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4–5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and i t was stable in the dark for several hours at –65°C.Four batho-intermediates were formed by irradiation with green light (500 nm) at –170°C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at –170°C. Four corresponding meta-intermediates were also formed by irradiation at –60°C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.