Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.     

Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIM_S, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.     

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor–3 (VEGFR–3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 μm in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31–positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR–3–positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE–1, Prox–1, neuropilin–2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE–1 were similar to those of VEGFR–3. Most lymphatic vessels were also identified by Prox–1. Neuropilin–2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR–3, LYVE–1, Prox–1, neuropilin–2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.     

Selective detection of 2-nitrobenzenesulfenyl-labeled peptides by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using a novel matrix

The 2-nitrobenzenesulfenyl (NBS) method, which is useful for quantitative proteome analysis, is based on stable isotope labeling of tryptophan residues with NBS chloride ((12)C(6)-NBSCl or (13)C(6)-NBSCl). We found that 3-hydroxy-4-nitrobenzoic acid (3H4NBA) is a more suitable matrix than 2,5-dihydroxybenzoic acid (DHB) for detecting NBS-labeled peptides by MALDI-quadrupole IT (QIT)-TOF MS . Furthermore, NBS-labeled peptides were selectively ionized and detected in a mixture of NBS-labeled and unlabeled peptides. Labeled paired peaks were easily detected without enrichment, nonpaired labeled peaks were clearly distinguished from unlabeled contaminating peptides, and nitrotyrosine-containing peptides were also selectively detected on the 3H4NBA matrix, while by-product-peaks arising from nitrobenzene moieties were suppressed. The use of 3H4NBA as a comatrix with CHCA improved the sensitivity of detection while substantially retaining the selectivity of 3H4NBA. The 3H4NBA matrix offers great advantages in terms of simplicity, sensitivity, and usability when used for the NBS method and for MALDI-TOF MS analysis applied to compounds having a nitrobenzene ring.     

Cytoplasmic inheritance of organelles in brown algae

Brown algae, together with diatoms and chrysophytes, are a member of the heterokonts. They have either a characteristic life cycle of diplohaplontic alternation of gametophytic and sporophytic generations that are isomorphic or heteromorphic, or a diplontic life cycle. Isogamy, anisogamy and oogamy have been recognized as the mode of sexual reproduction. Brown algae are the characteristic group having elaborated multicellular organization within the heterokonts. In this study, cytoplasmic inheritance of chloroplasts, mitochondria and centrioles was examined, with special focus on sexual reproduction and subsequent zygote development. In oogamy, chloroplasts and mitochondria are inherited maternally. In isogamy, chloroplasts in sporophyte cells are inherited biparentally (maternal or paternal); however, mitochondria (or mitochondrial DNA) derived from the female gamete only remained during zygote development after fertilization. Centrioles in zygotes are definitely derived from the male gamete, irrespective of the sexual reproduction pattern. Female centrioles in zygotes are selectively broken down within 1–2 h after fertilization. The remaining male centrioles play a crucial role as a part of the centrosome for microtubule organization, mitosis, determination of the cytokinetic plane and cytokinesis, as well as for maintaining multicellularity and regular morphogenesis in brown algae.     

Erratum to: Aromatic C–H bond hydroxylation by P450 peroxygenases: a facile colorimetric assay for monooxygenation activities of enzymes based on Russig’s blue formation

Aromatic C–H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450_BSβ (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig’s blue. Russig’s blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements.     

Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus.

In this paper, we report MALDI-TOF ms analysis of 2-nitrobenzenesulfenyl (NBS) labeled peptides with the powerful aid of an LC-automatic spotting system. using this approach we analyzed mammalian sera (rat and mouse) as biological samples to demonstrate performance. The labeling was carried out using a binary set of 2-nitrobenzenesulfenyl chloride (heavy and light), which modified tryptophan residues in sample proteins. Approximately 1600 doublet peaks were detected in the mass spectrum, some of which had more than threefold differences in their intensities. systematic separation/spotting followed by mass analysis of the NBS-labeled peptides derived from biological samples is described for the first time. This method has proved to be an effective application of NBS-labeled peptides and can be a powerful technique for quantitative analysis of proteins expressed in biological systems.     

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E^rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.