Isolation and characterization of hydrophobic proteins (H proteins) in the membrane fraction of Bacillus subtilis. Involvement in membrane biosynthesis and the formation of biochemically active membrane vesicles by combining H proteins with lipid.

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter. We named this H-protein. H-protein formed trilamellar unit membrane structure when combined with lipid. In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H-protein. However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein). Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid. H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid. Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems. Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.     

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases

Summary: Targeted mutagenesis of genes‐of‐interest, or gene‐knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN‐mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN‐mediated germ cell mutagenesis. genesis 52:431–439, 2014. © 2014 Wiley Periodicals, Inc.     

Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1

Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.