The anaphase promoting complex targeting subunit Ama1 links meiotic exit to cytokinesis during sporulation in Saccharomyces cerevisiae

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APC^Ama1 is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1Δ mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1Δ mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1Δ mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APC^Ama1 functions to coordinate the exit from meiosis II with cytokinesis.     

Structural and functional evolution of three cardiac natriuretic peptides

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

Clinical Performance of a New Soluble CD14-Subtype Immunochromatographic Test for Whole Blood Compared with Chemiluminescent Enzyme Immunoassay: Use of Quantitative Soluble CD14-Subtype Immunochromatographic Tests for the Diagnosis of Sepsis

We previously reported that a soluble CD14-subtype (sCD14-ST) immunochromatographic test (ICT) for plasma is more convenient than chemiluminescent enzyme immunoassay (CLEIA), but plasma separation makes bedside measurements difficult. We developed a new sCD14-ST ICT for whole blood and investigated whether quantitative determinations of sCD14-ST by ICT were useful for diagnosing sepsis and severe sepsis/septic shock. We studied 20 patients who fulfilled two or more systemic inflammatory response syndrome (SIRS) criteria and 32 patients who had been diagnosed with sepsis or severe sepsis/septic shock. Whole blood was collected on day 0 (on admission) and day 7, and the sCD14-ST concentration was quantitatively measured by CLEIA and ICT for whole blood. The patients’ Acute Physiology and Chronic Health Evaluation (APACHE) II, Sequential Organ Failure Assessment (SOFA), and Mortality in Emergency Department Sepsis (MEDS) scores were also calculated. The cut-off values obtained by the quantitative measurements made by ICT were 464.5 pg/mL for sepsis and 762.7 pg/mL for severe sepsis/septic shock (P < 0.0001). A Bland–Altman plot showed that no fixed bias or proportional bias was detected between CLEIA and quantitative ICT for whole blood. sCD14-ST concentrations were significantly correlated with APACHE II, SOFA, and MEDS scores (P < 0.0001). These results suggest that the new sCD14-ST ICT for whole blood may be a useful tool for the convenient, rapid bedside diagnosis and treatment of sepsis.     

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization,KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.     

Charge-pairing mechanism of phosphorylation effect upon amyloid fibrillation of human tau core peptide

Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer's disease. For elucidating molecular mechanisms of the phosphorylation effect on tau fibrillation, we synthesized a peptide, VQIVY 310K (PHF6) and its phosphorylated derivative (PHF6pY). PHF6 is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. To verify this charge-pairing mechanism, we conducted experiments using a series of PHF6 derivatives with non-native charge distributions. The electrostatic interaction in an intermolecular mode was also demonstrated by the system composed of two different peptide species, which found that fibrillation of nonphosphorylated PHF6 was drastically enhanced when a trace amount of phosphorylated PHF6 molecules coexisted. A simulation analysis utilizing crystal coordinates of the PHF6 fibril was also performed for interpreting the experimental results in a molecular level. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases.     

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.