Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica

The appendicularian, Oikopleura dioica, is a planktonic chordate. Its simple and transparent body, invariant cell lineages and short life cycle of 5 days make it a promising model organism for studies of chordate development. Here we describe the cell migration that occurs during development of the O. dioica larva. Using time-lapse imaging facilitated by florescent labeling of cells, three cell populations exhibiting long-distance migration were identified and characterized. These included (i) a multinucleated oral gland precursor that migrates anteriorly within the trunk region and eventually separates into the left and right sides, (ii) endodermal strand cells that are collectively retracted from the tail into the trunk in a tractor movement, and (iii) two subchordal cell precursors that individually migrate out from the trunk to the tip of the tail. The migration of subchordal cell precursors starts when all of the endodermal strand cells enter the trunk, and follows the same path but in a direction opposite to that of the latter. Labeling of these cells with a photoconvertible fluorescent protein, Kaede, demonstrated that the endodermal strand cells and subchordal cell precursors have distinct origins and eventual fates. Surgical removal of the trunk from the tail demonstrated that the endodermal strand cells do not require the trunk for migration, and that the subchordal cell precursors would be attracted by the distal part of the tail. This well-defined, invariant and traceable long-distance cell migration provides a unique experimental system for exploring the mechanisms of versatile cell migration in this simple organism with a chordate body plan.     

Membrane-microdomain localization of amyloid β-precursor protein (APP) C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668 residue

Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease.     

Inhibitory effect of SPE-39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF-stimulated cells

Although SPE-39 is a binding protein to Vps33B that is one of the subunit in the mammalian HOPS complex, the elements of SPE-39 function remain unknown. Here, we show that tyrosine phosphorylation of SPE-39 following EGF stimulation plays a role in the stability of SPE-39 itself. Ubiquitination of the C-terminal region of SPE-39 was also elevated in response to EGF stimulation, and this process was regulated by the phosphorylation of Tyr-11 in SPE-39. However, association of Vps33B with SPE-39 inhibited the elevation of ubiquitination of SPE-39 following EGF stimulation, which might be responsible for the stabilization of SPE-39. Furthermore, an opposing functional relationship between SPE-39 and Vps33B on the downregulation of the EGF receptor was observed in EGF-stimulated COS-7 cells.     

Simple fluorimetric method for quantification of sialic acids in glycoproteins

A simple and rapid fluorimetric method was developed for detection and quantitative analysis of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0 degrees C for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine derivative with acetoacetanilide and ammonia at room temperature for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, respectively. When the reaction was conducted in approximately a 1-ml volume, the linearity of the calibration exhibited between 2 and 180 microg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the standard deviation of the reagent blank, was 0.5 microg of fetuin. The proposed method was applied to determination of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extraction steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.     

Interaction of N-terminal acetyltransferase with the cytoplasmic domain of beta-amyloid precursor protein and its effect on A beta secretion

The processing of beta-amyloid precursor protein (APP) generates the amyloid beta-protein (A beta) and contributes to the development of Alzheimer's disease (AD). Elucidating the regulation of APP processing will, therefore, contribute to the understanding of AD. Many APP-binding proteins, such as FE65, X11s, and JNK-interacting proteins (JIPs), bind the motif 681-GYENPTY-687 within the cytoplasmic domain of APP. Here we found that the human homologue of yeast amino-terminal acetyltransferase ARD1 (hARD1) interacts with a novel motif, 658-HGVVEVD-664, in the cytoplasmic domain of APP695. hARD1 expressed its acetyltransferase activity in association with a human subunit homologous to another yeast amino-acetyltransferase, hNAT1. Co-expression of hARD1 and hNAT1 in cells suppressed A beta40 secretion and the suppression correlated with their enzyme activity. These observations suggest that the association of APP with hARD1 and hNAT1 and/or their N-acetyltransferase activity contributes to the regulation of A beta generation.     

Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity

The cellular protein retinoic acid-inducible gene I (RIG-I) senses intracellular viral infection and triggers a signal for innate antiviral responses including the production of type I IFN. RIG-I contains a domain that belongs to a DExD/H-box helicase family and exhibits an N-terminal caspase recruitment domain (CARD) homology. There are three genes encoding RIG-I-related proteins in human and mouse genomes. Melanoma differentiation associated gene 5 (MDA5), which consists of CARD and a helicase domain, functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with a helicase domain and transmit a signal downstream by CARD; thus, these proteins share overlapping functions. Another protein, LGP2, lacks the CARD homology and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5. The nonstructural protein 3/4A protein of hepatitis C virus blocks the signaling by RIG-I and MDA5; however, the V protein of the Sendai virus selectively abrogates the MDA5 function. These results highlight ingenious mechanisms for initiating antiviral innate immune responses and the action of virus-encoded inhibitors.     

Trace elements in human transitory milk

Multielement analysis was performed on human milk collected on 5-9-d postpartum from 51 Japanese females using inductively coupled plasma (ICP) mass spectrometry (MS), ICP atomic emission spectrometry (ICP-AES) and fluorometry. Thirty-one elements were detected by these analytical methods in milk. Twelve elements (Na, Mg, P, S, K, Ca, Cu, Zn, Se, Sr, Rb, and Mo) were detected in all of the samples. Al, Cs, and Ba were the elements detected by ICP-MS in more than half of the samples. Multiple regression analysis extracted biological attributes of mother and infant, such as maternal stature, maternal wt, or infant's birth wt, as statistically significant factors contributing to the variation in elemental concentration in milk. However, the rates of contribution were small in all cases. It was concluded that the biological attributes of mother and infant examined in this study were not the major factors that contribute to elemental variation in human milk.     

A novel type of kleptoplastidy in Dinophysis (Dinophyceae): presence of haptophyte-type plastid in Dinophysis mitra

Red-fluorescent, non-phycobilin-containing plastids were found in the heterotrophic dinoflagellate, Dinophysis mitra. Transmission electron microscopy showed that they contained a three-layer thylakoid, the absence of girdle lamella, and an embedded pyrenoid with thylakoid intrusions. These characteristics all coincide with haptophyte plastids. Phylogenetic analysis of the plastid small-subunit ribosomal DNA (SSU rDNA) revealed that the Dinophysis mitra sequences are distantly related to those of phycobilin-containing Dinophysis species and are positioned within a lineage of haptophytes belonging to Prymnesiophyceae. Because the plastid SSU rDNA sequences of Dinophysis mitra showed significant heterogeneity, despite being derived from a single species, it is highly likely that they were not established as plastids through an evolutionary process but are "kleptoplastids" (temporally stolen plastids) from multiple sources of haptophytes in the environment. We deduced that Dinophysis mitra takes up haptophytes myzocytotically and selectively retains the plastid with surrounding plastidal membranes, whereas other haptophyte cell components are degraded. This represents another type of kleptoplastidy in the Dinophysis species, which mostly harbor cryptophyte plastids, and is the first evidence of kleptoplastidy originating from haptophytes.     

Variations in plasma selenium levels as a result of the menstrual cycle and pregnancy in healthy Japanese women

Plasma levels of selenium (Se) were determined consecutively during a menstrual cycle of six women in three phases (i.e., menses, follicular, and luteal). To detect possible differences in relation to normal pregnancy, plasma levels of Se were also determined in paired samples of maternal and umbilical cord blood from 12 pregnant women. No periodic changes in the plasma Se levels were observed during the menstrual cycle. The intraindividual variation, estimated by coefficients of variation, ranged from 1.9% to 9.9% among the menstrual phases of the subjects. The plasma Se level during pregnancy did not differ significantly from those of nonpregnant women, and those in the second trimester and at delivery were at similar levels (1.58+/-.14 and 1.48+/-.20 mmol/L, respectively). Compared to the levels of maternal Se at delivery, the fetal cord plasma at birth had a significant lower Se level (1.23+/-.34 mmol/L, p<.05).     

Vacuolar Function in the Phosphate Homeostasis of the Yeast Saccharomyces cerevisiae

We studied physiological roles of the yeast vacuole in the phosphatemetabolism using ³¹P-in vivo nuclear magnetic resonance (NMR)spectroscopy. Under phosphate starvation wild-type yeast cellscontinued to grow for two to three generations, implying thatwild-type cells contain large phosphate pool to sustain thegrowth. During the first four hours under the phosphate starvedcondition, the cytosolic phosphate level was maintained almostconstant, while the vacuolar pool of phosphate decreased significantly.³¹P-NMR spectroscopy on the intact cells and perchloric acid(PCA) extracts showed that drastic decrease of polyphosphatetook place during this phase. In contrast,