Glycoprotein Ib distribution on the surface of platelets in resting and activation states: An electron microscope study

Summary Using a monoclonal antibody (TM60) against glycoprotein (GP) Ib, we determined immunocytochemically how GPIb is distributed on the platelet surface. When glutaraldehyde-fixed platelets were incubated with TM60, a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm. The number of ferritin particles on the surface of one side were 2070–4150 (2940 ± 790; mean ±s.d.,n = 10) under the scanning electron microscope. The distribution of ferritin particles was somewhat disarranged on the surface of unfixed platelets incubated with TM60 compared to that in the fixed platelets. Cluster-like structures of ferritin particles were observed in several places. When platelets were activated with ristocetin or thrombin, the distribution of ferritin particles was disturbed and cluster formation was observed in several places on the surface. These findings suggest that GPIb is uniformly distributed on the surface of platelets in the resting state, and that cluster formation occurs during activation of platelets.     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

Species-dependent migration of fish hatching gland cells that commonly express astacin-like proteases in common

Two constituent proteases of the hatching enzyme of the medaka ( Oryzias latipes ), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above-mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish ( Brachydanio rerio ) and masu salmon ( Oncorynchus masou ) embryos. Using the amplified fragments as probes, two full-length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.     

Effects of carp serum on the growth of goldfish fin cells in early passage

The culture medium supplemented with carp serum and fetal bovine serum (FBS) promoted cell growth significantly and induced morphological change of goldfish fin cells in early passage as compared to the medium containing FBS alone. However, these effects were not observed in RBCF-1, a cell line established from the goldfish fin. The sensitivity of the cells in early passage to carp serum suggests the following possibilities: (1) cells in early passage retain the ability to respond to growth-promoting factors specifically included in carp serum; and (2) this ability is lost during the process of long-term culture and/or long-term culture in FBS eliminates cell groups showing high dependency of cell growth on carp serum.     

Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with multiple eyespots

We have isolated a new Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with from one up to more than four eyespots cell^?1. It was designated mes (multiple eyespots)‐10 A wild‐type cell has a single eyespot, located under the chloroplast envelope, at a certain position near the cell's equator where the chloroplast envelope is in contact with the cell membrane. The eyespot(s) in mes‐10, however, are located at various positions on its chloroplast. The mes‐10 cells displayed negative phototaxis to 480–500 nm light. This behavior differed from that of a similar mutant, ptx4, which has been shown to have multiple eyespots and display no phototaxis (Pazour et al., J. Cell Biol. 1995; 131 : 427–40). Mes‐10 may retain a functional photoreceptor and a photosignal transduction system independently of its multiple eyespots. This mutant should be useful for studying how C. reinhardtii responds to light signals, as well as how eyespots are formed in the cell.     

Mycoplasma infection and hypoxia initiate succinate accumulation and release in the VM-M3 cancer cells

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1β production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.     

Alendronate suppresses tumor angiogenesis by inhibiting Rho activation of endothelial cells

We previously reported that alendronate inhibits intraperitoneal dissemination in an in vivo ovarian cancer model. Recently, nitrogen-containing bisphosphonates have been reported to have antiangiogenic activities. In this study, alendronate inhibited human umbilical vein endothelial cell (HUVEC) migration and capillary-like structure formation in vitro. These inhibitory effects were associated with reduced Rho activation and suppression of the formation of actin stress fibers and focal adhesions in HUVECs. Furthermore, the inhibition by alendronate was reversed by geranylgeraniol, which abrogated the inhibition of Rho geranylgeranylation. Next, we examined the effect of alendronate on angiogenesis in disseminated ovarian tumors of athymic immunodeficient mice. Alendronate treatment reduced the intra-tumor neoangiogenesis compared with that in the non-treated mice, although tumor-derived VEGF expression was not altered. In conclusion, the in vivo anti-tumor effect of alendronate might be derived, at least in part, from its direct antiangiogenic effects on intra-tumor endothelial cells by inhibiting Rho geranylgeranylation.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Synthesis and SAR studies of 3,6-disubstituted indazole derivatives as potent hepcidin production inhibitors

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of indazole compounds as hepcidin production inhibitors. The optimization study of compound 1 led to a potent hepcidin production inhibitor 45, which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.