The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.     

Structural study on the free lipid A isolated from lipopolysaccharide of Porphyromonas gingivalis.

The chemical structure of lipid A isolated from Porphyromonas gingivalis lipopolysaccharide was elucidated by compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The hydrophilic backbone of free lipid A was found to consisted of beta(1,6)-linked D-glucosamine disaccharide 1-phosphate. (R)-3-Hydroxy-15-methylhexadecanoic acid and (R)-3-hydroxyhexadecanoic acid are attached at positions 2 and 3 of the reducing terminal residue, respectively, and positions 2' and 3' of the nonreducing terminal unit are acylated with (R)-3-O-(hexadecanoyl)-15-methylhexadecanoic acid and (R)-3-hydroxy-13-methyltetradecanoic acid, respectively. The hydroxyl group at position 4' is partially replaced by another phosphate group, and the hydroxyl groups at positions 4 and 6' are unsubstituted. Considerable heterogeneity in the fatty acid chain length and the degree of acylation and phosphorylation was detected by liquid secondary ion-mass spectrometry (LSI-MS). A significant pseudomolecular ion of lipid A at m/z 1,769.6 [M-H]- corresponding to a diphosphorylated GlcN backbone bearing five acyl groups described above was detected in the negative mode of LSI-MS. Predominant ions, however, were observed at m/z 1,434.9 [M-H]- and m/z 1,449.0 [M-H]-, each representing monophosphoryl lipid A lacking (R)-3-hydroxyhexadecanoic and (R)-3-hydroxy-13-methyltetradecanoic acids, respectively. The presence of mono- and diphosphorylated lipid A species was also confirmed by LSI-MS of de-O-acylated lipid A (m/z 955.3 and 1,035.2, respectively).     

Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Prevalence of Cryptosporidium infection among domestic cats in the Tokyo Metropolitan District, Japan

From February, 1988 to February, 1989, an epidemiological survey for feline cryptosporidiosis was carried out on domestic cats that had been committed to the Tokyo Metropolitan Animal Protection Center by owners residing in the Tokyo Metropolitan District. Of a total of 608 cats, 23 (3.8%) were found to have Cryptosporidium oocysts. The percentage of cats positive for the infection ranged from 0 to 13.6% by month. The infection was found to be more common in "baby" and "young" cats than in "middle" and "old" ones. No significant difference in incidence was shown between the sexes. In each positive cat, no apparent clinical symptom such as diarrhea was found, and the size of the oocysts measured from 4.3 to 4.7 microns in diameter (small type).     

Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes.

Fission yeast Schizosaccharomyces pombe temperature-sensitive (ts) cut1 mutants fail to separate sister chromatids in anaphase but the cells continue to divide, leading to bisection of the undivided nucleus (the cut phenotype). If cytokinesis is blocked, replication continues, forming a giant nucleus with polyploid chromosomes. We show here that the phenotype of ts cut2-364 is highly similar to that of cut1 and that the functions of the gene products of cut1+ and cut2+ are closely interrelated. The cut1+ and cut2+ genes are essential for viability and interact genetically. Cut1 protein concentrates along the short spindle in metaphase as does Cut2. Cut1 (approximately 200 kDa) and Cut2 (42 kDa) associate, as shown by immunoprecipitation, and co-sediment as large complexes (30 and 40S) in sucrose gradient centrifugation. Their behavior in the cell cycle is strikingly different, however: Cut2 is degraded in anaphase by the same proteolytic machinery used for the destruction of cyclin B, whereas Cut1 exists throughout the cell cycle. The essential function of the Cut1-Cut2 complex which ensures sister chromatid separation may be regulated by Cut2 proteolysis. The C-terminal region of Cut1 is evolutionarily conserved and similar to that of budding yeast Esp1, filamentous fungi BimB and a human protein.     

The Role of the Phylogenetically Conserved Cochaperone Protein Droj2/DNAJA3 in NF-κB Signaling

The NF-κB pathway is a phylogenetically conserved signaling pathway with a central role in inflammatory and immune responses. Here we demonstrate that a cochaperone protein, Droj2/DNAJA3, is involved in the activation of canonical NF-κB signaling in flies and in human cultured cells. Overexpression of Droj2 induced the expression of an antimicrobial peptide in Drosophila. Conversely, Droj2 knockdown resulted in reduced expression of antimicrobial peptides and higher susceptibility to Gram-negative bacterial infection in flies. Similarly, Toll-like receptor-stimulated IκB phosphorylation and NF-κB activation were suppressed by DNAJA3 knockdown in HEK293 cells. IκB kinase overexpression-induced NF-κB phosphorylation was also compromised in DNAJA3 knockdown cells. Our study reveals a novel conserved regulator of the NF-κB pathway acting at the level of IκB phosphorylation.     

Genomic architecture of habitat‐related divergence and signature of directional selection in the body shapes of Gnathopogon fishes

Evolution of ecomorphologically relevant traits such as body shapes is important to colonize and persist in a novel environment. Habitat‐related adaptive divergence of these traits is therefore common among animals. We studied the genomic architecture of habitat‐related divergence in the body shape of Gnathopogon fishes, a novel example of lake–stream ecomorphological divergence, and tested for the action of directional selection on body shape differentiation. Compared to stream‐dwelling Gnathopogon elongatus, the sister species Gnathopogon caerulescens, exclusively inhabiting a large ancient lake, had an elongated body, increased proportion of the caudal region and small head, which would be advantageous in the limnetic environment. Using an F₂ interspecific cross between the two Gnathopogon species (195 individuals), quantitative trait locus (QTL) analysis with geometric morphometric quantification of body shape and restriction‐site associated DNA sequencing‐derived markers (1622 loci) identified 26 significant QTLs associated with the interspecific differences of body shape‐related traits. These QTLs had small to moderate effects, supporting polygenic inheritance of the body shape‐related traits. Each QTL was mostly located on different genomic regions, while colocalized QTLs were detected for some ecomorphologically relevant traits that are proxy of body and caudal peduncle depths, suggesting different degree of modularity among traits. The directions of the body shape QTLs were mostly consistent with the interspecific difference, and QTL sign test suggested a genetic signature of directional selection in the body shape divergence. Thus, we successfully elucidated the genomic architecture underlying the adaptive changes of the quantitative and complex morphological trait in a novel system.     

Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.     

Occurrence of O-phosphorylated 2-keto-3-deoxyoctonate in the lipopolysaccharide of Bacteroides gingivalis

Abstract Periodate-thiobarbituric acid reaction-positive substances were found in the strong acid hydrolysates of the lipopolysaccharide (LPS) from Bacteroides gingivalis 381. They were not identical to 2-keto-3-deoxyoctonate (KDO) in high-voltage paper electrophoresis (HVPE), their electrophoretic mobilities relative to KDO being 1.54 and 1.80, respectively. Alkaline phosphatase treatment and HVPE demonstrated that they are some kind of O-phosphorylated derivatives of KDO; in particular, the slow-moving component is identical, at least in HVPE, to 5- O -phosphoryl-KDO isolated from the strong acid hydrolysates of Bordetella pertussis (phase I) LPS.     

Ca2+ transients control CNS neuronal migration

In the developing CNS, postmitotic neurons exhibit dynamic changes in the mode, direction and rate of migration as they traverse different cortical layers, but the mechanisms underlying this process is largely unknown. Recent studies show that the changes in Ca2+ transient frequency play a central role in controlling the neuronal cell migration in a cortical layer-specific manner. In this article, we will first describe how granule cells migrate through different terrains of the developing cerebellar cortex. We will then present how such migration of granule cells is controlled by altering the Ca2+ transient frequency in their somata. Finally, we will discuss how the loss of Ca2+ transients triggers the completion of granule cell migration at their final destination.