Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4 ؉ T, CD8 ؉ T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.cell migration ͉ dendritic cells ͉ lymphocyte ͉ photoconvertible protein

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, we used the RNA silencing-interference technique (RNAi) to eliminate the Drosophila geminin homologue from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/ Grapes in the checkpoint control in response to overreplication. We found that the silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. We observed that the silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, our studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells.

Abstract:Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca 2ϩ uptake, intracellular Ca 2ϩ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10 Ϫ12 -10 Ϫ10 M) increased 45 Ca 2ϩ uptake and intracellular Ca 2ϩ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 M) or anti-EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5-day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10 Ϫ13 -10 Ϫ10 M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti-EPO antibody. Incubation with EPO (10 Ϫ13 -10 Ϫ10 M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10 Ϫ13 -10 Ϫ10 M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti-EPO antibody. Following a 4-h incubation with EPO (10 Ϫ14 -10 Ϫ10 M), NO production was increased, which was blunted by nicardipine and anti-EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca 2ϩ channels. Key Words: Erythropoietin-PC12 cells-Ca 2ϩ channels-Cell survivalDopamine release -Nitric oxide.

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Cohesive interactions between Porphyromonas gingivalis and plaque-forming bacteria, such as Streptococcus oralis, are considered to play an important role in the colonization of P. gingivalis in periodontal sites. Although P. gingivalis fimbriae have been reported to mediate coaggregation with S. oralis, the S. oralis molecule involved has not been identified. We identified the coadhesin of S. oralis ATCC 9811 and purified it by affinity column chromatography. We found that the molecular mass of the purified protein was approximately 40 kDa. Dot blot and Western blot assays showed binding of the 40-kDa protein to P. gingivalis fimbriae. Further, turbidimetric assays showed that the coadhesin inhibited coaggregation between P. gingivalis and S. oralis in a dose-dependent manner. Analyses of the amino-terminal sequences of the protein and its lysyl endopeptidase-cleaved fragments revealed that the coadhesin was identical to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, we cloned the gene that encodes S. oralis GAPDH and found that the sequence had a high degree of homology with the sequences of GAPDHs of various bacteria, including Streptococcus gordonii and Fusobacterium nucleatum. To confirm the contribution of S. oralis GAPDH to the interaction with P. gingivalis, a recombinant GAPDH protein was generated in Escherichia coli; this protein bound to P. gingivalis fimbriae and had an inhibitory effect on coaggregation. These results suggest that S. oralis GAPDH functions as a coadhesin for P. gingivalis fimbriae. In addition, considering the high degree of homology of the GAPDHs of various bacteria, those of other plaque-forming bacteria also may contribute to the colonization of P. gingivalis.Porphyromonas gingivalis is considered to be a prominent periodontopathogen, as it possesses a variety of virulence factors, such as proteinases, hemagglutinin, and lipopolysaccharide (28,37,41). In order to cause infection, it is necessary for P. gingivalis to attach to tooth surfaces, subgingival epithelium, or early colonizing gram-positive bacteria; this step constitutes the initial stage of colonization in periodontal pockets (38). In addition, the bacterium has been reported to interact with a variety of other oral gram-positive bacteria (19), including Actinomyces naeslundii (36, 43), Actinomyces viscosus (9, 12, 26, 27, 36), Streptococcus gordonii (23), Streptococcus mutans (17), Streptococcus oralis (29), and Streptococcus sanguis (39); these interactions are considered to play a vital role in the colonization of P. gingivalis in the oral cavity.P. gingivalis possesses several cell surface components that are important for its attachment. Its fimbriae have been shown to interact with epithelial cells (15), cultured human fibroblasts (20), and saliva-coated hydroxyapatite beads (2, 32), while its vesicles have been shown to interact with collagen-coated hydroxyapatite beads (32). Further, in studies to determine which of the surface components of P. gingivalis interact with grampositive bacteria, its fimbri...

Methyl esters from crude sunflower oil were produced via methanolysis reaction using sodium hydroxide catalyst. Methanolysis was carried out at different agitation speeds (200-600 rpm), temperatures (25-60• C), catalyst loadings (0.25-1.00% by weight of oil), and methanol:oil mole ratios (6:1-20:1). Mass-transfer limitation was effectively minimized at agitation speeds of 400-600 rpm with no apparent lag period. Lowering the temperature resulted in a fall in the rate of reaction prolonging the reaction time necessary to achieve maximum production of methyl ester. Using 0.50% hydroxide catalyst was found to be adequate, resulting in 97-98% conversion without compromising recovery due to soap formation. Increasing the methanol:oil mole ratio beyond the usual amount of 6:1 tended to speed up the initial rate of methanolysis and was found to lower the bonded glycerol content, especially the amount of diglyceride in the sample. Kinetic rate constants were derived from experimental results using second-order rate expressions, and values of activation energy for glyceride methanolysis have been established.

We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17beta-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor.

Interactions of the type 1 phosphatase catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MBP) were compared using affinity columns. In the absence of ATP, MBP bound to dephosphorylated myosin, heavy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not bound. In the presence of ATP, the binding of MBP occurred only with phosphorylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitive inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K(m) for the latter. With myosin, MBP showed a marked increase in kcat, compared to PP1c. ATP did not affect these parameters. To investigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were expressed. These activated PP1c, and activation was maximum at approximately an equimolar ratio. The equimolar mixture of recombinant fragment and PP1c exhibited K(m) values similar to MBP and increased kcat values, compared to PP1c alone. An affinity column was prepared using the recombinant fragment. Phosphorylated HMM and P-LC20 were bound in the presence and absence of ATP. The interaction of P-LC20 was not ATP-dependent. Dephosphorylated HMM did not bind in the presence of ATP. The N-terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the large subunit of MBP contained binding sites for P-LC20 and PP1c.