Synthesis and biological evaluation of deoxy salacinols, the role of polar substituents in the side chain on the alpha-glucosidase inhibitory activity

Three analogs (5, 6, and 7) lacking polar substituents in the side chain of a naturally occurring alpha-glucosidase inhibitor, salacinol (1a), were synthesized by the coupling reaction of a thiosugar, 1,4-dideoxy-1,4-epithio-D-arabinitol (3), with cyclic sulfates (8, 9, and 10), and their alpha-glucosidase inhibitory activities were examined. All these simpler analogs (5, 6, and 7) showed less inhibitory activity compared to 1a, and proved the importance of cooperative role of the polar substituents for the alpha-glucosidase inhibitory activity. A practical synthetic route to 3 starting from D-xylose is also described.     

Arg interacts with cortactin to promote adhesion-dependent cell edge protrusion

The molecular mechanisms by which the Abelson (Abl) or Abl-related gene (Arg) kinases interface with the actin polymerization machinery to promote cell edge protrusions during cell–matrix adhesion are unclear. In this study, we show that interactions between Arg and the Arp2/3 complex regulator cortactin are essential to mediate actin-based cell edge protrusion during fibroblast adhesion to fibronectin. Arg-deficient and cortactin knockdown fibroblasts exhibit similar defects in adhesion-dependent cell edge protrusion, which can be restored via reexpression of Arg and cortactin. Arg interacts with cortactin via both binding and catalytic events. The cortactin Src homology (SH) 3 domain binds to a Pro-rich motif in the Arg C terminus. Arg mediates adhesion-dependent phosphorylation of cortactin, creating an additional binding site for the Arg SH2 domain. Mutation of residues that mediate Arg–cortactin interactions abrogate the abilities of both proteins to support protrusions, and the Nck adapter, which binds phosphocortactin, is also required. These results demonstrate that interactions between Arg, cortactin, and Nck1 are critical to promote adhesion-dependent cell edge protrusions.     

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.     

Efficient production of offspring from Japanese wild-derived strains of mice (Mus musculus molossinus) by improved assisted reproductive technologies

Because the genetic diversity of the laboratory mouse (Mus musculus) is very limited, wild-derived strains from this genus could provide invaluable experimental models for studies of mouse genetics and epigenetics such as quantitative trait locus analysis. However, such strains generally show poor reproductive performance under conventional husbandry conditions, so their use for large-scale analyses has been limited. This study was undertaken to devise assisted reproductive technologies (ARTs) for the efficient production of offspring in two wild-derived strains, MSM/Ms and JF1/Ms (Mus musculus molossinus). First, as females of these strains are poor responders to equine chorionic gonadotropin (eCG) stimulation, we examined the efficiency of superovulation by injecting anti-inhibin serum followed by human chorionic gonadotropin (hCG). Approximately four to six times more oocytes were ovulated than with eCG-hCG treatment in both strains, reaching ～25-30 oocytes per female. Consequently, the procedures for in vitro fertilization using these superovulated oocytes and cryopreservation of embryos and spermatozoa could be optimized for both of the wild-derived strains. However, MSM/Ms embryos but not JF1/Ms embryos failed to develop to term after embryo transfer because of intrauterine death at mid to late gestation. We were able to overcome this obstacle by cotransfer of these embryos with those from laboratory strains combined with treatment of recipient females with an immunosuppressant (cyclosporin A). Thus, a series of ARTs essential for efficient production and preservation of the wild-derived strains were successfully devised. These technologies will facilitate systematic studies of mouse genetics and epigenetics using a wider range of genetic diversity than currently available in the genus Mus.     

Distribution and histologic effects of intravenously administered amorphous nanosilica particles in the testes of mice

Amorphous nanosilica particles (nSP) are being utilized in an increasing number of applications such as medicine, cosmetics, and foods. The reduction of the particle size to the nanoscale not only provides benefits to diverse scientific fields but also poses potential risks. Several reports have described the in vivo and in vitro toxicity of nSP, but few studies have examined their effects on the male reproductive system. The aim of this study was to evaluate the testicular distribution and histologic effects of systemically administered nSP. Mice were injected intravenously with nSP with diameters of 70 nm (nSP70) or conventional microsilica particles with diameters of 300 nm (nSP300) on two consecutive days. The intratesticular distribution of these particles 24h after the second injection was analyzed by transmission electron microscopy. nSP70 were detected within sertoli cells and spermatocytes, including in the nuclei of spermatocytes. No nSP300 were observed in the testis. Next, mice were injected intravenously with 0.4 or 0.8 mg nSP70 every other day for a total of four administrations. Testes were harvested 48 h and 1 week after the last injection and stained with hematoxylin-eosin for histologic analysis. Histologic findings in the testes of nSP70-treated mice did not differ from those of control mice. Taken together, our results suggest that nSP70 can penetrate the blood-testis barrier and the nuclear membranes of spermatocytes without producing apparent testicular injury.     

The Drosophila miR-310 cluster negatively regulates synaptic strength at the neuromuscular junction

Emerging data implicate microRNAs (miRNAs) in the regulation of synaptic structure and function, but we know little about their role in the regulation of neurotransmission in presynaptic neurons. Here, we demonstrate that the miR-310-313 cluster is required for normal synaptic transmission at the Drosophila larval neuromuscular junction. Loss of miR-310-313 cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of mir-310-313 in larval presynaptic neurons. Kinesin family member, Khc-73 is a functional target for miR-310-313 as its expression is increased in mir-310-313 mutants and reducing it restores normal synaptic function. Cluster mutants show an increase in the active zone protein Bruchpilot accompanied by an increase in electron dense T bars. Finally, we show that repression of Khc-73 by miR-310-313 cluster influences the establishment of normal synaptic homeostasis. Our findings establish a role for miRNAs in the regulation of neurotransmitter release.     

Conversion of quinate to 3-dehydroshikimate by Ca-alginate-immobilized membrane of Gluconobacter oxydans IFO 3244 and subsequent asymmetric reduction of 3-dehydroshikimate to shikimate by immobilized cytoplasmic NADP-shikimate dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

In-advance trans-medullary stimulation of bone marrow enhances spontaneous repair of full-thickness articular cartilage defects in rabbits

Mesenchymal stromal cells (MSCs), especially those lying close to cartilage defects, are an important cell source for cartilage regeneration. We hypothesize that a larger number of MSCs might become available, if the bone marrow in the immediate vicinity of the subchondral bone is stimulated for MSCs in advance of the creation of cartilage defects. A trans-medullary passage-way reaching the immediate vicinity of the subchondral bone was created 4 days prior to the creation of cartilage defects. In another setting, basic fibroblast growth factor (bFGF) was administered through the trans-medullary passage-way in order to augment the stimulation of MSCs. The rabbits were killed at various times after the creation of cartilage defects. Triple staining of bromodeoxyuridine (BrdU), CD44 and CD45 and histological evaluation were subsequently performed. A considerable proportion of the proliferating cells were identified as bone-marrow-derived MSCs. Enumeration of BrdU-positive cells demonstrated that trans-medullary stimulation, especially with bFGF, increased the number of proliferating cells. The histological grading score of trans-medullary stimulation with bFGF group was superior to that of the other groups. Thus, in-advance stimulation of the bone marrow effectively increases the number of proliferating cells. The putative progenitor cells for chondrocytes stimulated thereby are likely to be recruited to the osteochondral defects at the appropriate time, contributing to the repair of full-thickness articular cartilage defects at the early follow-up time point.     

Spectroscopy on the B850 band of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila. I. Experiments and Monte Carlo simulations

The electronic structure of the circular aggregate of 18 bacteriochlorophyll a (BChl a) molecules responsible for the B850 absorption band of the light-harvesting 2 (LH2) complex of the photosynthetic purple bacterium Rhodopseudomonas acidophila has been studied by measuring fluorescence-excitation spectra of individual complexes at 1.2 K. The spectra reveal several well-resolved bands that are obscured in the single, broad B850 band observed in conventional absorption measurements on bulk samples. They are interpreted consistently in terms of the exciton model for the circular aggregate of BChl a molecules. From the energy separation between the different exciton transitions a reliable value of the intermolecular interaction is obtained. The spectra of the individual complexes allow for a distinction between the intra- and the intercomplex disorder. In addition to the random disorder, a regular modulation of the interaction has to be assumed to account for all the features of the observed spectra. This modulation has a C(2) symmetry, which strongly suggests a structural deformation of the ring into an ellipse.     

Dopamine induces IL-6-dependent IL-17 production via D1-like receptor on CD4 naive T cells and D1-like receptor antagonist SCH-23390 inhibits cartilage destruction in a human rheumatoid arthritis/SCID mouse chimera model

A major neurotransmitter dopamine transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1-D5. Several studies have shown that dopamine not only mediates interactions into the nervous system, but can contribute to the modulation of immunity via receptors expressed on immune cells. We have previously shown an autocrine/paracrine release of dopamine by dendritic cells (DCs) during Ag presentation to naive CD4(+) T cells and found efficacious results of a D1-like receptor antagonist SCH-23390 in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis and in the NOD mouse model of type I diabetes, with inhibition of Th17 response. This study aimed to assess the role of dopaminergic signaling in Th17-mediated immune responses and in the pathogenesis of rheumatoid arthritis (RA). In human naive CD4(+) T cells, dopamine increased IL-6-dependent IL-17 production via D1-like receptors, in response to anti-CD3 plus anti-CD28 mAb. Furthermore, dopamine was localized with DCs in the synovial tissue of RA patients and significantly increased in RA synovial fluid. In the RA synovial/SCID mouse chimera model, although a selective D2-like receptor antagonist haloperidol significantly induced accumulation of IL-6(+) and IL-17(+) T cells with exacerbated cartilage destruction, SCH-23390 strongly suppressed these responses. Taken together, these findings indicate that dopamine released by DCs induces IL-6-Th17 axis and causes aggravation of synovial inflammation of RA, which is the first time, to our knowledge, that actual evidence has shown the pathological relevance of dopaminergic signaling with RA.