Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.     

Enumeration of Legionella CFU by colony hybridization using specific DNA probes

Oligonucleotide probes and colony hybridization (CH) were applied to enumerate organisms of the genus Legionella in cooling tower water. The CH counts indicated almost the same results as CFU counts in cultivated samples derived from the water. It was concluded that it is possible to substitute the CH procedure for the conventional one.     

1H, 15N and 13C resonance assignments of yeast Saccharomyces cerevisiae calmodulin in the Ca2+-free state

Calmodulin (CaM) is a small Ca²⁺-binding protein, which has been found in all of eucaryotic cells examined. CaMs isolated from various species have highly conserved amino acid sequence (more than 90% identical), and show the same biological functions. CaM isolated from the baker's yeast (Saccharomyces cerevisiae) (yCaM), however, shares only 60% identity in the amino acid sequence with CaM from vertebrate, and shows quite distinct conformational and biochemical properties compared with those of CaM from other species. The conformational details of yCaM, however, have not been revealed yet. We achieved the chemical shift assignments of yCaM (146 amino acids) in the apo-state using uniformly ¹⁵N- and ¹³C-labeled protein. Consequently, the resonances of 95% atoms in the backbone amides were successfully assigned.     

Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.     

Regulation by organic acids of polysaccharide-mediated microbe-plant interactions

A polysaccharide flocculant of Klebsiella pneumoniae H12 has been suggested to mediate microbe-plant interactions with the aid of Ca2+ [K. Nakata et al., Biosci. Biotechnol. Biochem., 64, 459-465, 2000]. Here, two-way regulation of polysaccharide-mediated interactions between K. pneumoniae and Raphanus sativus was studied using organic acids. Namely, 10 mM equivalents of organic acids promoted production of the polysaccharide by the bacterium, but inhibited flocculation of bacterial cells by the polysaccharide. These phenomena were counterbalanced by equi-molar equivalents of Ca2+, suggesting competition for Ca2+ between the carboxylic residues of the polysaccharide and those of the aliphatic acids. By electron microscopy observations, bacterial cell aggregates were sparsely distributed over the main roots and root hairs, had various sizes, and seemed to tightly adhere to root tissues. Their shapes seemed to be distorted and abundant in cavities. In brief, these microscopical observations may be explained by a two-way regulation system of bacterial adhesion to a plant by organic acids.     

Engagement of 4-1BB inhibits the development of experimental allergic conjunctivitis in mice

The 4-1BB receptor acts as a costimulator in CD8(+) T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-gamma production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-gamma knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-gamma.     

Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex

Vertebrate Wnt proteins activate several distinct pathways. Intrinsic differences among Wnt ligands and Frizzled (Fzd) receptors, and the availability of pathway-specific coreceptors, LRP5/6, and Ror2, affect pathway selection. Here, we show that a secreted glycoprotein, Cthrc1, is involved in selective activation of the planar cell polarity (PCP) pathway by Wnt proteins. Although Cthrc1 null mutant mice appeared normal, the introduction of a heterozygous mutation of a PCP gene, Vangl2, resulted in abnormalities characteristic of PCP mutants. In HEK293T cells, Cthrc1 activated the PCP pathway but suppressed the canonical pathway. Cell-surface-anchored Cthrc1 bound to Wnt proteins, Fzd proteins, and Ror2 and enhanced the interaction of Wnt proteins and Fzd/Ror2 by forming the Cthrc1-Wnt-Fzd/Ror2 complex. Consistent with this, Ror2 mutant mice also showed PCP-related abnormalities in the inner ear. These results suggest that Cthrc1 is a Wnt cofactor protein that selectively activates the Wnt/PCP pathway by stabilizing ligand-receptor interaction.     

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.     

A fatal case of fungal endocarditis of the tricuspid valve associated with long-term venous catheterization and treatment with antibiotics in a patient with a history of alcohol abuse

We report a fatal case of fungal (candidal) endocarditis of the tricuspid valve with clinico-pathologically interesting findings following and associated with candidal pneumonia during long-term central venous catheterization (CVC) for intravenous therapy and long-term treatment with antibiotics for bacterial and fungal infection in a patient with a history of alcohol abuse. We review the literature on fungal cardiac infection related to long-term catheterization and alcohol abuse, and discuss the pathogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.