Characterization of monoclonal antibodies against human parvovirus B19

Eleven hybridoma cell lines producing mouse monoclonal antibodies (mAbs) against human parvovirus B19 were established. Their specificity was as follows. Approximately 5% of fetal erythroid cells inoculated with B19 reacted with all the mAbs and with anti-B19 positive human serum, but not with negative serum by indirect double immunofluorescence staining. All the mAbs recognized both VP-1 (84 kDa) and VP-2 (58 kDa) capsid proteins of B19 virions propagated in vitro and in vivo by Western blotting, and immunoprecipitated B19 virions.     

Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

Uterine leiomyoma, also known as fibroids, is the most common benign neoplasm of the female genital tract. Leiomyoma is the most common uterine tumor. The leiomyoma subtypes account for approximately 10% of leiomyomas. Intravenous leiomyomatosis, a uterine leiomyoma subtype, is an intravascular growth of benign smooth muscle cells, occasionally with pelvic or extrapelvic extension. Uterine leiomyosarcoma, a malignant tumor, tends to metastasize hematogenously, and distant metastasis to the lungs and liver is common. Therefore, the oncological properties of this intravenous leiomyomatosis resemble those of the malignant tumor uterine leiomyosarcoma. Cancer stem cells migrate to distant organs via intravascular infiltration, leading to micrometastases. We examined the oncological properties of intravenous leiomyomatosis using molecular pathological techniques on tissue excised from patients with uterine leiomyoma. CD44-positive mesenchymal tumor stem-like cells were detected in both patients with intravenous leiomyomatosis and uterine leiomyosarcoma. The oncological properties of intravenous leiomyomatosis were found to be similar to those of uterine leiomyosarcoma. However, in intravenous leiomyomatosis, cyclin E and Ki-67-positive cells were rare and no pathological findings suspecting malignancy were observed. It is expected that establishing a treatment method targeting cancer stem cells will lead to the treatment of malignant tumors with a low risk of recurrence and metastasis.     

The ASB2β Ubiquitin-interacting motif is involved in its monoubiquitination

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2β. ASB2β has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2β but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2β and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2β protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2β, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2β is produced in cells. ASB2β with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2β M1) or a Ser38Ala mutation, (ASB2β M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2β monoubiquitination. Furthermore, an ASB2β mutant that lacked a SOCS box (ASB2β ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2β. In contrast, an ASB2β mutant that lacked the UIM-containing domain (ASB2β ΔN) was not monoubiquitinated. These results suggest that ASB2β but not ASB2α might be monoubiquitinated and that the ASB2β UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination.     

Vascular endothelial cells promote cortical neurite outgrowth via an integrin β3-dependent mechanism

The interaction of neurons with their non-neuronal milieu plays a crucial role in the formation of neural networks, and wide variety of cell-contact-dependent signals that promote neurite elongation have been identified. In this study, we found that vascular endothelial cells promote neurite elongation in an integrin β3-dependent manner. Vascular endothelial cells from the cerebral cortex promoted neurite elongation of cortical neurons in a cell contact-dependent manner. This effect was mediated by arginine–glycine–aspartic acid (RGD), a major recognition sequence for integrins. Pharmacological blockade of integrin β3 abolished the neurite elongation effect induced by the endothelial cells. Immunocytochemical analysis revealed that integrin β3 was expressed on cultured cortical neurons. These results demonstrate that the neurite elongation promoted by vascular endothelial cells requires integrin β3. Vascular endothelial cells may therefore play a role in the development and repair of neural networks in the central nervous system.     

Circadian regulation of bioluminescence in the prey-luring glowworm, Arachnocampa flava

The glowworms of New Zealand and Australia are bioluminescent fly larvae that generate light to attract prey into their webs. Some species inhabit the constant darkness of caves as well as the dim, natural photophase of rain-forests. Given the diversity of light regimens experienced by glowworms in their natural environment, true circadian rhythmicity of light output could be present. Consequently the light emission characteristics of the Australian subtropical species Arachnocampa flava, both in their natural rainforest habitat and in artificial conditions in the laboratory, were established. Larvae were taken from rainforest and kept alive in individual containers. When placed in constant darkness (DD) in the laboratory they maintained free-running, cyclical light output for at least 28 days, indicating that light output is regulated by an endogenous rhythm. The characteristics of the light emission changed in DD: individuals showed an increase in the time spent glowing per day and a reduction in the maximum light output. Most individuals show a free-running period greater than 24 h. Manipulation of the photophase and exposure to skeleton photoperiods showed that light acts as both a masking and an entraining agent and suggests that the underlying circadian rhythm is sinusoidal in the absence of light-based masking. Manipulation of thermoperiod in DD showed that temperature cycles are an alternative entraining agent. Exposure to a period of daily feeding in DD failed to entrain the rhythm in the laboratory. The endogenous regulation of luminescence poses questions about periodicity and synchronization of bioluminescence in cave glowworms.     

Generation of a syngeneic mouse model to study the intraperitoneal dissemination of ovarian cancer with in vivo luciferase imaging

In order to facilitate the discovery and investigation of anti‐cancer therapeutics under physiological conditions, we have engineered the ovarian cancer cell line, HM‐1/luc, in mice. This cell stably expresses firefly luciferase and produces light that can be detected using an in vivo imaging system (IVIS). Parental HM‐1 cells cause severe carcinomatous peritonitis to B6C3F1 mice, but not to C57BL6 mice. Established HM‐1/luc cells showed pathologically similar findings to HM‐1 cells. HM‐1/luc cells were injected into the peritoneal cavity of B6C3F1 mice and IVIS 2000 was conducted weekly after inoculation to monitor intra‐peritoneal tumor growth. The mice were divided into three groups: non‐CDDP‐treated (control) and CDDP‐treated (0.2 and 0.4 mg). A disease‐suppressive effect of the CDDP was reflected by the significantly prolonged survival of the CDDP‐treated mice (control 23 ± 1.9 days, CDDP 0.2 mg 29.6 ± 2.9 days; p < 0.05); the total photon and area of flux were decreased. The optical imaging of intraperitoneal tumors via in vivo bioluminescence is effective for noninvasive monitoring and semi‐quantitative analysis. Our syngeneic mouse model has the relevant clinical features of ovarian cancer, which makes it a useful model for developing new ovarian cancer therapies. Copyright © 2009 John Wiley & Sons, Ltd.     

Two sides of lifespan regulating genes: pro-longevity or anti-longevity?

Traditionally, ageing has been considered a passive and entropic process, in which damages accumulate on biological macromolecules over time and the accumulated damages lead to a decline in overall physiological functions. However, the discovery of a longevity mutant in the nematode Caenorhabditis elegans has challenged this view. A longevity mutant is a mutant organism, in which a reduction-of-function of a certain gene prolongs the lifespan. Thus, the discovery of longevity mutants has shown the existence of genes, which function to shorten lifespan in wild-type organisms, promoting extensive hunting for longevity-regulating genes in short-lived model organisms, such as yeast, worms and flies. These studies have revealed remarkable conservation of longevity-regulating genes and their networks among species. Decreased insulin/IGF-like signalling and decreased target of rapamycin (TOR) signalling are both shown to extend lifespan in evolutionarily divergent species, from unicellular organisms to mammals. Intriguingly, most of these longevity-regulating pathways reveal pro-longevity and anti-longevity effects on lifespan, depending on biological and environmental contexts. This review summarizes pleiotropic functions of the conserved longevity-regulating genes or pathways, focusing on studies in C. elegans.     

Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

Herbicide resistance of transgenic rice plants expressing human CYP1A1

Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.