Internalization of a novel,huge lectin from <Emphasis Type="Italic">Ibacus novemdentatus</Emphasis> (slipper lobster) induces apoptosis of mammalian cancer cells

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and ?3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.     

Determinants of early initiation of breastfeeding in rural Niger: cross-sectional study of community based child healthcare promotion

Background

Most child deaths are preventable and caused by behaviorally modifiable factors. By promoting optimal breastfeeding, we can reduce neonatal and child mortality risks by 45%. This paper provides new family and community based perspectives to identify factors interfering with the program impact on promoting early initiation of breastfeeding among the most vulnerable populations in rural Niger.

Methods

A secondary analysis of a retrospective cross-sectional study evaluated a UNICEF behavior change program on child healthcare. The study sample is based on a post-hoc constitution of two groups exposed and unexposed to the program. All women (n = 1026) aged 14–49 years having at least one child below 24 months of age were included. We measured crude and adjusted odds ratios with chi-square and multivariate logistic regression models.

Results

Independent variables shown to be associated with early breastfeeding include sales activities compared to household work with no direct income (AOR 7.7; 95% CI 1.3, 47.8) and mutual decision for harvest use (AOR 8.6; 95% CI 2.0, 36.8). Antenatal care did not modify the timing of breastfeeding initiation.

Conclusions

A high risk group of mothers with social and economic vulnerability are prone to suboptimal breastfeeding within the first hour of birth. Support from family and neighbors positively influenced early breastfeeding. Those who had no direct income and limited access to health services were a high-risk group, prone to delayed initiation of breastfeeding.

     

Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.     

Differential usage of COX-1 and COX-2 in prostaglandin production by mast cells and basophils

Basophils have been erroneously considered as minor relatives of mast cells, due to some phenotypic similarity between them. While recent studies have revealed non-redundant roles for basophils in various immune responses, basophil-derived effector molecules, including lipid mediators, remain poorly characterized, compared to mast cell-derived ones. Here we analyzed and compared eicosanoids produced by mouse basophils and mast cells when stimulated with IgE plus allergens. The production of 5-LOX metabolites such as LTB4 and 5-HETE was detected as early as 0.5 h post-stimulation in both cell types, even though their amounts were much smaller in basophils than in mast cells. In contrast, basophils and mast cells showed distinct time course in the production of COX metabolites, including PGD2, PGE2 and 11-HETE. Their production by mast cells was detected at both 0.5 and 6 h post-stimulation while that by basophils was detectable only at 6 h. Of note, mast cells showed 8–9 times higher levels of COX-1 than did basophils at the resting status. In contrast to unaltered COX-1 expression with or without stimulation, COX-2 expression was up-regulated in both cell types upon activation. Importantly, when activated, basophils expressed 4–5 times higher levels of COX-2 than did mast cells. In accordance with these findings, the late-phase production of the COX metabolites by basophils was completely ablated by COX-2 inhibitor whereas the early-phase production by mast cells was blocked by COX-1 but not COX-2 inhibitor. Thus, the production of COX metabolites is differentially regulated by COX-1 and COX-2 in basophils and mast cells.     

Regulation of the anaphase-promoting complex-separase cascade by transforming growth factor-beta modulates mitotic progression in bone marrow stromal cells

Alteration of the tumor microenvironment by aberrant stromal cells influences many aspects of cell biology, including differentiation of stem cells and tumor metastasis. The role of transforming growth factor (TGF)-β signaling in stromal cells of the tissue microenvironment is critical to both pathways. We examined murine marrow stromal cells with deletion of Smad3 and found that they have an altered cell cycle profile, with a higher fraction of cells in G2/M phase. Deletion of Smad3 significantly abrogates TGF-β signaling and suppresses phosphorylation of CDC27–anaphase-promoting complex (APC) during mitosis, thereby resulting in elevated cyclin-dependent kinase (CDK)1 activity via increased levels of cyclin B. Enhanced CDK1 activity due to deregulation of APC leads in turn to hyperphosphorylation of separase, impeding chromatid separation. A residue Ser1126Ala mutation in separase specifically abolished separase hyperphosphorylation in Smad3-deficient cells. The present results unveil a new function for the TGF-β pathway in the regulation of APC to mediate chromatid separation during mitosis.     

An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

Molecular cloning of urea transporters from the kidneys of baleen and toothed whales

Urea transport in the kidney is important for the production of concentrated urine. This process is mediated by urea transporters (UTs) encoded by two genes, UT-A (Slc14a2) and UT-B (Slc14a1). Our previous study demonstrated that cetaceans produce highly concentrated urine than terrestrial mammals, and that baleen whales showed higher concentrations of urinary urea than sperm whales. Therefore, we hypothesized that cetaceans have unique actions of UTs to maintain fluid homeostasis in marine habitat. Kidney samples of common minke (Balaenoptera acutorostrata), sei (B. borealis), Bryde's (B. brydei) and sperm whales (Physeter macrocephalus) were obtained to determine the nucleotide sequences of mRNAs encoding UT. The sequences of 2.5-kb cDNAs encode 397-amino acid proteins, which are 90-94% identical to the mammalian UT-A2s. Two putative glycosylation sites are conserved between the whales and the terrestrial mammals, whereas consensus sites for protein kinases are not completely conserved; only a single protein kinase A consensus site was identified in the whale UT-A2s. Two protein kinase C consensus sites are present in the baleen whale UT-A2s, however, a single protein kinase C consensus site was identified in the sperm whale UT-A2. These different phosphorylation sites of whale UT-A2s may result in the high concentrations of urinary urea in whales, by reflecting their urea permeability.