Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

Acid-induced changes in the in vivo wall-yielding properties of hypocotyl sections of Vigna unguiculata

Elongation growth of hypocotyl sections of Vigna unguiculata under xylem perfusion was significantly enhanced when acid was applied by acid-aerosol to an abraded hypocotyl surface in the air. The in vivo wall extensibility (φ) and the effective turgor (Pⁱ– Y), both of which were determined by the pressure-jump method, increased during acid-induced growth as observed in IAA-induced growth. The intracellular pressure (Pⁱ), however, decreased significantly at the beginning of acid-induced growth whereas Pⁱ scarcely changed in IAA-induced growth. This result indicates that protons increase the effective turgor by decreasing the yield threshold as IAA does. There seems to be no essential difference between proton and auxin in the effects on the in vivo mechanical properties of the surface cell wall.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Role of a novel coiled-coil domain-containing protein CCDC69 in regulating central spindle assembly

The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.Key words: CCDC69, aurora B, Plk1, central spindles, midzone components, cytokinesis     

Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization

MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2(-/-)) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2(-/-)map1b(-/-) mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an "inside-out" pattern. Detailed observation of primary cultures of hippocampal neurons from map2(-/-)map1b(-/-) mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2(-/-)map1b(-/-) mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.     

Interleukin-1β Induces Substance P Release from Primary Afferent Neurons Through the Cyclooxygenase-2 System

Substance P (SP) is synthesized in the dorsal root ganglion (DRG) and released from primary afferent neurons to convey information regarding noxious stimuli. The effects of the proinflammatory cytokine interleukin-1 (IL-1) beta on the release of SP were investigated using primary cultured rat DRG cells. Recombinant mouse IL-1beta added to the cells at 0.1 ng/ml increased the SP-like immunoreactivity (SPLI) in the culture medium after incubation for 6 h by approximately 50% as compared with that of nontreated DRG cells. The effect of IL-1beta was Ca(2+)-dependent and significantly inhibited by 100 ng/ml IL-1 receptor-specific antagonist (IL-1r antagonist), cyclooxygenase (COX) inhibitors such as 0.1 mM aspirin, 1 microg/ml indomethacin, and 1 microM NS-398 (specific for COX-2), and 1 microM dexamethasone. Furthermore, a 1-h incubation with IL-1beta markedly increased the inducible COX-2 mRNA level, which was inhibited by an IL-1r antagonist and dexamethasone, whereas IL-1beta showed no effect on the level of constitutive COX-1 mRNA. These observations indicated that IL-1beta induced the release of SP from the DRG cells via specific IL-1 receptors, the mechanism of which might involve prostanoid systems produced by COX-2. This could be responsible for the hyperalgesic action with reference to inflammatory pain in the primary afferent neuron to spinal cord pathway.     

Cutting Edge: Plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ

Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.     

Distal Sox binding elements of the alphaB-crystallin gene show lens enhancer activity in transgenic mouse embryos

alphaB-Crystallin, a member of the small heat shock protein (sHSP) family, is expressed in various tissues including lens, heart, and skeletal muscle. Previously we identified the gene of HSPB2, another member of the sHSP family, located 1-kb upstream of the alphaB-crystallin gene in a head-to-head manner. In the present study, we found a highly conserved region of 220 bp approximately 2.4-kb upstream of the alphaB-crystallin gene and examined its role in expression of the alphaB-crystallin gene. Transgenic mice containing 3 kb of the upstream sequence of the alphaB-crystallin gene showed lacZ reporter gene expression in the lens as well as the myotome and heart on embryonic day 12.5. Deletion analysis revealed that the -2656/-2267 region including the conserved region with four putative Sox binding elements (E1-E4) exhibits lens enhancer activity toward the alphaB-crystallin promoter. Gel shift assays showed that the Sox1 and Sox2 proteins preferentially bound to E2 and E4. Moreover, disruption of E2 and E4 abolished the reporter gene expression in the lens. These results indicate that the newly identified enhancer with Sox elements activates the alphaB-crystallin promoter in the lens, although they are separated by the entire HSPB2 gene.     

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.