DNA stable pentaploid H1 (ES) cells obtained from an octaploid cell induced from tetraploid cells polyploidized using demecolcine

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one‐cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G₁, S, and G₂/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone‐like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long‐term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden. J. Cell. Physiol. 223: 369–375, 2010. © 2010 Wiley‐Liss, Inc.     

PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P₂], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P₂ production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P₂ is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.     

Pulmonary carcinosarcoma showing an obvious response to pazopanib: a case report

Background

Pulmonary carcinosarcoma (PCS) is a rare primary lung malignancy and has a poor prognosis among lung tumor histological subtypes. However, an appropriate treatment strategy has not been developed for unresectable PCS.

Case presentation

A 65-year-old man who was diagnosed with PCS was treated by surgical removal of the primary lung lesion, followed by six cycles of adjuvant chemotherapy with cisplatin plus irinotecan. Following the chemotherapy, he experienced a relapse with brain metastasis, which induced the rapid onset of left leg paralysis. Radical surgical resection and stereotactic radiosurgery to the resection cavity were performed. However, meningeal dissemination and new lung metastases occurred after a year and half. To control these multiple metastatic lesions, the patient was treated with the multiple kinase inhibitor pazopanib. No change was observed in the meningeal dissemination, while the metastatic lung lesions were prominently reduced in size following treatment with pazopanib. Consequently, the patient showed a partial response to pazopanib treatment, although the dose of pazopanib was reduced by half as a result of thrombocytopenia.

Conclusion

This is the first report of metastatic PCS showing an evident therapeutic response to tumor-targeted therapy. We suggest that pazopanib may be a therapeutic option for patients with metastatic PCS.

     

Alkaline adaptation induces cross-protection against some environmental stresses and morphological change in Vibrio parahaemolyticus

The relationship between alkaline adaptation and the resistance against environmental stresses was examined in Vibrio parahaemolyticus. Alkali-adapted cells were found to have increased resistance against various stresses, including heat, crystal violet, deoxycholic acid, and hydrogen peroxide. However, alkali-adapted cells showed no increased resistance against acid stress and heat-adapted cells did not show increased resistance against alkaline stress. Furthermore, alkaline treatment induced cell elongation with heterogenous size of the bacterium.     

Comparison of the physiology, morphology, and leaf demography of tropical saplings with different crown shapes

Branch architecture, leaf photosynthetic traits, and leaf demography were investigated in saplings of two woody species, Homolanthus caloneurus and Macaranga rostulata, co-occurring in the understory of a tropical mountain forest. M. rostulata saplings have cylindrical crowns, whereas H. caloneurus saplings have flat crowns. Saplings of the two species were found not to differ in area-based photosynthetic traits and in average light conditions in the understory of the studied site, but they do differ in internode length, leaf emergence rate, leaf lifespan, and total leaf area. Displayed leaf area of H. caloneurus saplings, which have the more rapid leaf emergence, was smaller than that of M. rostulata saplings, which have a longer leaf lifespan and larger total leaf area, although M. rostulata saplings showed a higher degree of leaf overlap. Short leaf lifespan and consequent small total leaf area would be linked to leaf overlap avoidance in the densely packed flat H. caloneurus crown. In contrast, M. rostulata saplings maintained a large total leaf area by producing leaves with a long leaf lifespan. In these understory saplings with a different crown architecture, we observed two contrasting adaptation strategies to shade which are achieved by adjusting a suite of morphological and leaf demographic characters. Each understory species has a suite of morphological traits and leaf demography specific to its architecture, thus attaining leaf overlap avoidance or large total leaf area.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation

Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.     

Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice

Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.