INTERMEDIATE FEATURES OF CYANELLE DIVISION OF CYANOPHORA PARADOXA (GLAUCOCYSTOPHYTA) BETWEEN CYANOBACTERIAL AND PLASTID DIVISION1

Cyanelles of glaucocystophytes may be the most primitive of the known plastids based on their peptidoglycan content and the sequence phylogeny of cyanelle DNA. In this study, EM observations have been made to characterize the cyanelle division of Cyanophora paradoxa Korshikov and to gain insights into the evolution of plastid division. Constriction of cyanelles involves ingrowth of the septum at the cleavage site with the inner envelope membrane invaginating at the leading edge and the outer envelope membrane invaginating behind the septum. This means the inner and outer envelope membranes do not constrict simultaneously as they do in plastid division in other plants. The septum and the cyanelle envelope became stained after a silver‐methenamine staining was applied for in situ detection of polysaccharides. Septum formation was inhibited by β‐lactams and vancomycin, which are potent inhibitors of bacterial peptidoglycan biosynthesis. These results suggest the presence of peptidoglycan at the septum and the cyanelle envelope. In dividing cyanelles, a single electron‐dense ring (cyanelle ring) was observed on the stromal face of the inner envelope membrane at the isthmus, but no ring‐like structures were detected on the outer envelope membrane. Thus a single, stromal cyanelle ring such as this is quite unique and also distinct from FtsZ rings, which are not detectable by TEM. These features suggest that the cyanelle division of glaucocystophytes represents an intermediate stage between cyanobacterial and plastid division. If monophyly of all plastids is true, the cyanelle ring and the homologous inner plastid dividing ring might have evolved earlier than the outer plastid dividing ring.     

Persistent Release of IL-1s from Skin Is Associated with Systemic Cardio-Vascular Disease,Emaciation and Systemic Amyloidosis: The Potential of Anti-IL-1 Therapy for Systemic Inflammatory Diseases

The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1β antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.     

The amino-terminal half of Sendai virus C protein is not responsible for either counteracting the antiviral action of interferons or down-regulating viral RNA synthesis

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.     

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.     

Resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-infected T-cell lines

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-kappaB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-kappaB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-lLnorleucinal (LLnL), an NF-kappaB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-kappaB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-kappaB may be a useful adjunct in clinical use of Apo2L against ATL.     

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti‐ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C‐terminal src kinase)‐binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent‐resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti‐ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over‐expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314‐phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314‐phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP‐43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.     

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54^−/−/KU70^−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54^−/−/LIG4^−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.     

Autologous Bone-Marrow Mesenchymal Stem Cell Implantation and Endothelial Function in a Rabbit Ischemic Limb Model

Background

The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.

Methods

We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10⁶ MSCs were implanted into each ischemic limb.

Results

Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N^G-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.

Conclusions

These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.     

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model

BACKGROUND: A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. METHODS: We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. CONCLUSIONS: Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections.