Novel identification of the different types of cones in the retina of the chicken

1. Although single cones and double cones in the chicken retina had been documented for more than 30 years, the exact morphology of these cells had never been studied by the scanning electronmicroscopy. In this brief report, we present the evidence for the first time the existence of two types of single cones and three types of double cones (termed as types A, B, and C), each with distinct morphology.2. The proportion of type A:type B:type C double cones, as estimated from the midperipheral and central regions of our scanning specimens, was about 30%:50%:20%.3. Based on the literature data that red oil droplets reside in single cones and yellow oil droplets in chief cones of the double cones, the proportion of single to double cones was deciphered and the relative proportion was estimated to be 1:0.91 in the central region, 1:0.92 in the midperiphery, and 1:0.84 in the periphery.     

Expression,purification, and crystallization of glutamyl-tRNA(Gln) specific amidotransferase from Bacillus stearothermophilus

Although the genes that encode the glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) specific amidotransferase (Glu-AdTase) from various bacteria and eukaryotic organelles are known, the precise mechanism of the enzyme is still unclear. One of the reasons is that there is no information on the three-dimensional structure of the complex, the Glu-AdTase:Glu-tRNA(Gln):ATP:amino group donor. To obtain the crystals of Glu-AdTase, the Glu-AdTase of Bacillus stearothermophilus was overexpressed and purified after cloning of the gene that encodes the enzyme. The cloned DNA contained the full-length gene cluster that represented the Glu-AdTase of B. stearothermophilus, and was organized as an operon that consisted of three open-reading frames (ORFs). The order of the genes was gatCAB, as shown in Bacillus subtilis. The ORFs showed a high amino-acid homology to those of B. subtilis (A subunit, 73.2%; B subunit, 81.6%; C subunit, 69.5%) and Staphylococcus aureus (A subunit, 61.9%; B subunit, 71.8%; C subunit, 45.9%). The ORFs were re-cloned on the overexpression vector, pTrc99a, and a recombinant pTrcgatCABBST was obtained. The Glu-AdTase that was overexpressed with pTrcgatCABBST in Escherichia coli retained transamidation activity on the mischarged glutamic acid on the tRNA(Gln). It also produced correctly-charged Gln-tRNA(Gln) at 37, 42, and 50 degrees C. Although Glu-AdTases from both B. subtilis and B. stearothermophilus were subjected to crystallization, the micro-crystals were only obtained from the B. stearothermophilus enzyme.     

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.     

Toxoplasma gondii-derived heat shock protein 70 induces lethal anaphylactic reaction through activation of cytosolic phospholipase A2 and platelet-activating factor via Toll-like receptor 4/myeloid differentiation factor 88

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce IFN-gamma-dependent lethal anaphylactic reaction in T. gondii-infected mice through an alternative PAF-mediated pathway, but not the classical immunoglobulin (Ig)E-dependent pathway. Although marked IFN-gamma production was observed by CD11b(+), CD11c(+), CD4(+) and CD8(+) splenocytes, CD11b(+) and CD11c(+) cells were shown to be the key effecter cells which generated pro-inflammatory lipid such as PAF and caused T.g.HSP70-induced anaphylactic reaction. In the present study, we found that the T.g.HSP70-induced anaphylactic reaction was not observed in TLR 4-deficient ((-/-)) mice, whereas it was observed in WT and TLR2(-/-) mice. The mRNA expression of PAF-AH, the main enzyme for PAF degradation, increased in T. gondii-infected WT and TLR2(-/-) but not in TLR4(-/-) mice after T.g.HSP70 injection. Furthermore, phosphorylation of cPLA(2), which is the key enzyme for pro-inflammatory lipid generation, was detected in CD11b(+) splenocytes of WT and TLR2(-/-) mice but not in TLR4(-/-) mice. Subsequently, cPLA(2) activation was suppressed by inhibiting the TLR4-directed p38 and p44/42 MAPK pathways. However, T.g.HSP70-induced anaphylactic reaction was observed in TRIF(-/-) mice, but not in MyD88(-/-) mice. These findings indicate the cPLA(2) activated-PAF production via TLR4/MyD88-dependent, but not TRIF-dependent, signaling pathway in T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice.     

Ciglitizone inhibits cell proliferation in human uterine leiomyoma via activation of store-operated Ca2+ channels

This study investigated the acute effects of a peroxisome proliferator-activated receptor (PPAR)- ligand, ciglitizone, on cell proliferation and intracellular Ca²⁺ signaling in human normal myometrium and uterine leiomyoma. Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured with fura-2 AM, and cellular viabilities were determined by viable cell count and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay. Ciglitizone (100 µM) induced greater inhibition of cell proliferation in uterine leiomyoma than in myometrium. Ciglitizone also dose-dependently increased [Ca²⁺]_i in both myometrium and uterine leiomyoma; these [Ca²⁺]_i increases were inhibited by PPAR- antagonists and raloxifene. Ciglitizone-induced [Ca²⁺]_i increase showed only an initial peak in normal myometrial cells, whereas in uterine leiomyoma there was a second sustained [Ca²⁺]_i increase as well. The initial [Ca²⁺]_i increase in both myometrium and uterine leiomyoma resulted from the release of Ca²⁺ by the sarcoplasmic reticulum via activation of ryanodine receptors. The second [Ca²⁺]_i increase was observed only in uterine leiomyoma because of a Ca²⁺ influx via an activation of store-operated Ca²⁺ channels (SOCCs). Cell proliferation was inhibited and secondary [Ca²⁺]_i increase in uterine leiomyoma was attenuated by cotreatment of ciglitizone with a SOCC blocker, lanthanum. The results suggest that ciglitizone inhibits cell proliferation and increases [Ca²⁺]_i through the activation of SOCCs, especially in human uterine leiomyoma. peroxisome proliferator-activated receptor-; intracellular calcium; uterine cells     

Bax-dependent apoptosis induced by ceramide in HL-60 cells

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. In this study, we show that antisense bax inhibits cytochrome c release, poly(ADP-ribose)polymerase cleavage and cell death induced by ceramide in HL-60 cells. In addition, ceramide induces translocation of Bax to mitochondria. The addition of the broad spectrum caspase inhibitor zVAD-fmk prevented ceramide-induced apoptotic cell death but did not inhibit translocation of Bax and mitochondrial cytochrome c release. Furthermore, ceramide inhibits the expression of the antiapoptotic protein Bcl-xL with an increase in the ratio of Bax to Bcl-xL. These data provide direct evidence that Bax plays an important role in regulating ceramide-induced apoptosis.     

Analysis of the Genetic Diversity in the Vigna minima Complex and Related Species in East Asia

Vigna subgenus Ceratotropis (V. angularis, V. minima, V. nakashimae, V. reflexo-pilosa, V. riukiuensis and V. umbellata) was investigated using AFLP methodology. Species in the V. minima complex, V. minima, V. nakashimae and V. riukiuensis, had greater intra-specific polymorphism than the other species analyzed. Vigna species from East Asia analysed could be clearly distinguished. The relationship between species was consistent using different analyses and showed V. riukiuensis and V. nakashimae are closely related. The phenetic distance between species (1-Jaccard's similarity coefficient) ranged from 0.279 between V. riukiuensis and V. nakashimae to 0.688 between V. reflexo-pilosa and V. minima. Genetic distance based on inferred nucleotide diversity (π) ranged from 0.012 between V. riukiuensis and V. nakashimae and 0.053 between both V. riukiuensis and V. nakashimae and V. reflexo-pilosa. The diversity (Ha) within species, based on Shannon's information index of phenotypic diversity, was lowest in V. reflexo-pilosa (0.006) and highest in V. minima (0.095). Korean and Japanese populations of V. nakashimae had distinct polymorphism. Vigna riukiuensis populations from Miyako island were genetically different from populations of other islands of southern Okinawa. Received 21 April 2000/ Accepted in revised form 5 September 2000     

Comparative sequence and methylation analysis of chloroplast and amyloplast genomes from rice

Plant Molecular Biology - Grain amyloplast and leaf chloroplast DNA sequences are identical in rice plants but are differentially methylated. The leaf chloroplast DNA becomes more methylated as the...