Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin

The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

Cardiolipin induces premature senescence in normal human fibroblasts

Lipids seem to have various roles in cellular senescence. We found that cardiolipin very sensitively inhibits growth of normal human fibroblasts, whereas other phospholipids do not at 100 times higher concentrations. Growth arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. Senescence markers such as the p21(waf1/sdi-1), fibronectin, and collagenase-I genes were significantly upregulated by cardiolipin. In addition, caldiolipin significantly increased in normally senesced human fibroblasts leaving other phospholipids unaltered. These results suggest that accumulation of cardiolipin is one of the causes for replicative senescence.     

Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells.

BCR/ABL tyrosine kinase generated from the chromosomal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To examine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (Delta p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or Delta p85; that of cyclin E by N17; and that of cyclin A by Delta p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Delta p85 were hardly effective. In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or Delta p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-alpha- and dexamethasone-induced apoptosis, disruption of one pathway by N17, 694F, or Delta p85 sensitized K562 to these reagents. These results suggested that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.     

Phosphorylation of the amino-terminal region of X11L regulates its interaction with APP

X11-like (X11L) is neuronal adaptor protein that interacts with the amyloid β-protein precursor (APP) and regulates its metabolism. The phosphotyrosine interaction/binding (PI/PTB) domain of X11L interacts with the cytoplasmic region of APP695. We found that X11L–APP interaction is enhanced in osmotically stressed cells and X11L modification is required for the enhancement. Amino acids 221–250 (X11L^221–250) are required for the enhanced association with APP in osmotically stressed cells; this motif is 118 amino acids closer to the amino-terminal end of the protein than the PI/PTB domain (amino acids 368–555). We identified two phosphorylatable seryl residues, Ser236 and Ser238, in X11L^221–250 and alanyl substitution of either seryl residue diminished the enhanced association with APP. In brain Ser238 was found to be phosphorylated and phosphorylation of X11L was required for the interaction of X11L and APP. Both seryl residues in X11L^221–250 are conserved in neuronal X11, but not in X11L2, a non-neuronal X11 family member that did not exhibit enhanced APP association in osmotically stressed cells. These findings indicate that the region of X11L that regulates association with APP is located outside of, and amino-terminal to, the PI/PTB domain. Modification of this regulatory region may alter the conformation of the PI/PTB domain to modulate APP binding.     

cDNA cloning and expression of rat tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor for the factor VIIa-tissue factor complex. We isolated cDNA for rat TFPI by screening a lambda gt10 rat liver cDNA library. We determined the 1,228 bp nucleotide sequence, comprising a 88 bp 5' non-coding region, a 906 bp open reading frame, and a 234 bp 3' non-coding region, which encodes a protein of 302 amino acid residues. On Northern blot analysis of rat TFPI mRNA, rat TFPI mRNA was detected as two forms with different molecular sizes, 4.0 and 1.4 kb, which were expressed abundantly in heart, lung, kidney, and aortic endothelial cells. The homology of the amino acid sequence of rat TFPI with those of human and rabbit TFPI was found to be 60.7 and 57.4%, respectively. The lengths of the three tandem Kunitz-type inhibitor domains were strictly conserved not only among TFPI from the three species, but also among other proteins containing Kunitz-type inhibitor domains. The homology of the Kunitz-type domains in TFPI among the three species was 57, 86, and 69% in the 1st, 2nd, and 3rd domains, respectively. There was no significant difference in hydropathy profiles of TFPI from man, rabbit, and rat.     

Analysis of microbiota involved in the aged natural fermentation of indigo

Although the indigo reduction process is performed via natural fermentation and maintained under open-air condition, the indigo-reducing reactions continue for 6 months (on average) or longer. Identifying the mechanism underlying the maintenance of this process could lead to the development of a novel, long-lasting, unsterilized bioprocesses. To determine the mechanisms underlying the maintenance of the indigo fermentation system microbiota for more than 6 months in a reduced state in an anaerobic alkaline environment, we examined changes in the microbiota in one early-phase batch and two aged batches of indigo fermentation fluid. The microbiota in the aged fermentation fluid consisted mainly of the genera Alkalibacterium, Amphibacillus, Anaerobacillus and Polygonibacillus and the family Proteinivoraceae. The genera Alkalibacterium, Amphibacillus and Polygonibacillus are known to include indigo-reducing bacteria. Although the transition speed was slower in the aged fermentation fluid than in the early-stage fluid, the microbiota in the aged fermentation fluid maintained for more than 6 months was drastically changed within a period of 3 months. The results of this study indicate that the bacterial consortia consisted of various indigo-reducing species that replace the previous group of indigo-reducing bacteria. The notable transitional changes may be concomitant with changes in the environmental conditions, such as the nutritional conditions, observed over 3 months. This flexibility may lead to important changes in the microbiota that allow for the maintenance of a fermentation-reducing state over a long period.     

Establishment of a new initial dose plan for vancomycin using the generalized linear mixed model

Background

When administering vancomycin hydrochloride (VCM), the initial dose is adjusted to ensure that the steady-state trough value (Css-trough) remains within the effective concentration range. However, the Css-trough (population mean method predicted value [PMMPV]) calculated using the population mean method (PMM) often deviate from the effective concentration range. In this study, we used the generalized linear mixed model (GLMM) for initial dose planning to create a model that accurately predicts Css-trough, and subsequently assessed its prediction accuracy.

Methods

The study included 46 subjects whose trough values were measured after receiving VCM. We calculated the Css-trough (Bayesian estimate predicted value [BEPV]) from the Bayesian estimates of trough values. Using the patients’ medical data, we created models that predict the BEPV and selected the model with minimum information criterion (GLMM best model). We then calculated the Css-trough (GLMMPV) from the GLMM best model and compared the BEPV correlation with GLMMPV and with PMMPV.

Results

The GLMM best model was {[0.977?+?(males: 0.029 or females: -0.081)]?×?PMMPV?+?0.101?×?BUN/adjusted SCr – 12.899?×?SCr adjusted amount}. The coefficients of determination for BEPV/GLMMPV and BEPV/PMMPV were 0.623 and 0.513, respectively.

Conclusion

We demonstrated that the GLMM best model was more accurate in predicting the Css-trough than the PMM.

     

Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and related compounds

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 microg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by (31)P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.     

Genealogical analysis of subspecies divergence and spikelet-shape diversification in central Eurasian wild wheat Aegilops tauschii Coss.

The genealogical and geographic structure of variation in spikelet morphology was analyzed for central Eurasian wild wheat Aegilops tauschii Coss. using a diverse array of 203 sample accessions that represented the entire species range. In this sample set, two subspecies were identified on the basis of sensu-stricto criteria: only the accessions having markedly moniliform spikes were assigned to Ae. tauschii Coss. subspecies strangulata (Eig) Tzvel., whereas those having mildly moniliform and cylindrical spikes to Ae. tauschii Coss. subspecies tauschii. In a graph of the first two axes from a principal component analysis based on nine spikelet traits, the plots of the two subspecies formed separate clusters, indicating that subspecies strangulata sens. str. is a practically usable taxon. Chloroplast-DNA-based genealogical analyses suggested that subspecies strangulata diverged from an ancestor that carried a specific chloroplast DNA type, whereas, after divergence, this subspecies became polyphyletic, likely through hybridization. Geographically, significant longitudinal and latitudinal clines were detected for spikelet size, with spikelets tending to be small in the eastern and southern regions. These results shed some light on the patterns of subspecies divergence and spikelet-shape diversification in the course of Ae. tauschii’s long-distance dispersal from the Transcaucasus to China.