Bromovirus movement protein genes play a crucial role in host specificity.

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small.     

Tocopherols play a crucial role in low-temperature adaptation and Phloem loading in Arabidopsis

To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.     

Defensins play a crucial role in protecting mice against oral Shigella flexneri infection

An earlier study revealed that 4-day-old mice, but not older mice, were infected with invasive Shigella strains. Here we attempted to determine the underlying mechanism that induces inflammation in the intestines of neonate mice after oral Shigella infection. Wild-type BALB/c mice of different ages (7, 14, and 35 days old) were orally administered GFP-expressing Shigella flexneri 5a M90T strain (5 × 10⁹ CFU) and analyzed for colonization 6 h following infection. We found that Shigella localized in the epithelium, lamina propria, and crypt regions of the small intestines of 7-day-old BALB/c mice. Microarray analysis revealed that expression levels of cryptdin and various types of cryptdin-related mRNA (e.g., cryptrs-2, -5, -7, -12 and lysozyme) in the small intestines were significantly lower in 7-day-old than in older mice regardless of Shigella infection status. Interestingly, matrix metalloprotease-7 (matrilysin)-deficient (MAT^−/−) mice of B6 background had more colonies and more severe symptoms of inflammation in the intestines than did wild-type B6 mice after oral Shigella challenge. This suggests that cryptdin-related antimicrobial molecules are indispensable for efficient protection against oral Shigella infection.     

The role of complement activation in tumour necrosis factor-induced lethal hepatitis.

Injection of tumour necrosis factor (TNF) in animals causes severe liver cell toxicity, especially when D-(+)-galactosamine (GalN) is co-administered. After challenge with TNF/GalN, serum complement activity (CH50 and APCH50) decreased dramatically, suggesting strong activation of both the classical and the alternative pathways. TNF or GalN alone had no such effect. A cleavage product of complement protein C3 [C3(b)] was deposited on the surface of hepatocytes of TNF/GalN-treated mice. Intravenous administration of cobra venom factor (CVF), which depletes complement, inhibited the development of hepatitis. However, CVF pretreatment also protected C3-deficient mice. Pretreatment of mice with a C1q-depleting antibody did not prevent TNF/GalN lethality, although the anti-C1q antibody had depleted plasma C1q. Factor B-deficient and C3-deficient mice, generated by gene targeting, proved to be as sensitive to TNF/GalN as control mice. Furthermore, induction of lethal shock by platelet-activating factor, an important mediator in TNF-induced hepatic failure, was not reduced in C3-deficient mice. These data indicate that complement, although activated, plays no major role in the generation of acute lethal hepatic failure in this model and that CVF-induced protection is independent of complement depletion.     

Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis

We recently identified a minireplicon of pBtoxis from Bacillus thuringiensis subsp. israelensis that contained an operon encoding two novel proteins (ORF156 and ORF157), both of which are required for replication. ORF157 contains a helix-turn-helix motif and shares no homology with known plasmid replication proteins (Rep), and ORF156 contains the signature motif present in FtsZ/tubulin proteins, the latter of which are known to function in cell division and chromosome segregation. Here we show that the minimal sequence composed of four 12-bp imperfect direct repeats (iterons) in the pBtoxis minireplicon was sufficient to replicate a reporter plasmid in B. thuringiensis subsp. israelensis when ORF156 and ORF157 functions were provided in trans. To further investigate the roles of ORF156 and ORF157 in pBtoxis replication, six-histidine-tagged recombinant rORF156 and rORF157 proteins were purified from Escherichia coli and used in electrophoretic mobility shift assays. Our results demonstrated that rORF157, but not rORF156, binds specifically to the pBtoxis iterons, suggesting that ORF157 functions as a Rep protein. Although rORF156 did not bind to the iteron sequence, we showed that it bound to rORF157-DNA complexes. In addition, we showed that rORF156 has GTPase activity characteristic of the FtsZ/tubulin superfamily of proteins. Taken together, these results suggest that the iterons compose the minimal replication origin (ori) of pBtoxis and that ORF157 and ORF156 are involved in the initiation of pBtoxis replication and possibly in the segregation and partitioning of this plasmid to daughter cells.     

Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica

Entamoeba histolytica is the human parasite responsible of amoebiasis, during which highly motile trophozoites invade the intestinal epithelium leading to amoebic colitis, and disseminate via the blood circulation causing liver abscesses. The invasive process, central to the pathogenesis, is known to be driven by parasites motility. To investigate molecules responsible for in vivo motion, we performed a high resolution dynamic imaging analysis using two-photon laser scanning microscopy. Image analysis of the parasites during invasion of Caco-2 cell monolayers, an enterocyte-like model, and hamster liver shows that E. histolytica undergoes non-Brownian motion. However, studies of movements of parasite strains dominant negative for myosin II, a central component of the cytoskeleton, and for Gal-GalNAc lectin, a major adhesion molecule, indicate that myosin II is essential for E. histolytica intercellular motility through intestinal cell monolayers and for its motility in liver. In contrast, the Gal-GalNAc lectin exclusively triggers invasion of the liver. These observations are in agreement with emerging studies that highlight marked differences in the way that cells migrate in vitro in two dimensions versus in vivo in three dimensions. The approach that we have developed should be powerful to identify adhesive complexes required for in vivo cell migration in normal and pathogenic situations and may, thereby, lead to new therapeutic drug, for pathologies based on cell motility and adhesion.     

Membrane rafts play a crucial role in receptor activator of nuclear factor kappaB signaling and osteoclast function

Membrane lipid rafts play a key role in immune cell activation by recruiting and excluding specific signaling components of immune cell surface receptors upon the receptor engagement. Despite this, the role of these microdomains in the regulation of osteoclasts as controlled by receptor activator of nuclear factor kappaB (RANK) has yet to be established. In this study, we demonstrate that the raft microdomain expression plays an essential role in osteoclast function and differentiation. Expression of raft component flotillin greatly increased during osteoclast differentiation, whereas engagement of RANK induced the translocation of tumor necrosis factor receptor-associated factor 6 to rafts where Src was constitutively resident. Disruption of rafts blocked TRAF6 translocation and Akt activation by RANK ligand in osteoclasts and further reduced the survival of osteoclasts. Actin ring formation and bone resorption by osteoclasts were also found to require the integrity of rafts. Our observations demonstrate for the first time that RANK-mediated signaling and osteoclast function are critically dependent on the expression and integrity of raft membrane microdomains.     

Nuclear aggregates of polyamines are supramolecular structures that play a crucial role in genomic DNA protection and conformation

In a previous study we showed that natural polyamines interact in the nuclear environment with phosphate groups to form molecular aggregates [nuclear aggregates of polyamines (NAPs)] with estimated molecular mass values of 8000, 4800 and 1000 Da. NAPs were found to interact with genomic DNA, influence its conformation and interfere with the action of nucleases. In the present work, we demonstrated that NAPs protect naked genomic DNA from DNase I, whereas natural polyamines (spermine, spermidine and putrescine) fail to do so. In the context of DNA protection, NAPs induced noticeable changes in DNA conformation, which were revealed by temperature-dependent modifications of DNA electrophoretic properties. In addition, we presented, for NAPs, a structural model of polyamine aggregation into macropolycyclic compounds. We believe that NAPs are the sole biological forms by which polyamines efficiently protect genomic DNA against DNase I, while maintaining its dynamic structure.     

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.     

EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.     

Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome

Objective

This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development.

Methods

The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB.

Results

The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlation between these proteins and GRP78.

Conclusions

Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications.     

Does boron play only a structural role in the growing tissues of higher plants?

In species in which boron (B) mobility is limited, B deficiency only occurs in growing plant organs. As a consequence of the highly localized patterns of plant growth and the general immobility of B it has been extremely difficult to determine the primary function of B in plants. In species in which B is phloem mobile, the removal of B from the growth medium results in the depletion of B present in mature leaves. Thus, it is possible to develop mature leaves with increasingly severe levels of B depletion, thereby overcoming the complications of experiments based on growing tissues. Utilizing this approach we demonstrate here that B depletion of mature plum (Prunus salicina) leaves did not result in any discernible change in leaf appearance, membrane integrity or photosynthetic capacity even though B concentrations were reduced to 6-8 µg/g dwt, which is less than 30% of the reported tissue B requirement. Boron depletion, however, results in a severe disruption of plant growth and metabolism in young growing tissues. This experimental evidence and theoretical considerations suggest that the primary and possibly sole function of B, is as a structural component of growing tissues.     

The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 10⁸ viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.     

Site-selective homing of antigen-primed lymphocyte populations can play a crucial role in the efferent limb of cell-mediated immune responses in vivo

Lymphoid cells of the mammalian immune system exhibit special migratory properties within their in vivo environment. This fundamental characteristic is important to the protection of the organism from infection and neoplastic transformation by a process termed immunologic surveillance. The importance of lymphocyte recirculation was addressed by investigating the role of site-selective homing of lymphocytes during the efferent phase of an in vivo adoptively transferred immune response. We approached this problem by using pertussis toxin (PT), a known inhibitor of lymphoid cell migration in vivo. PT is a protein secreted by the bacterium Bordetella pertussis, which induces a selective and long-lasting inhibition of the emigration of lymphocytes from the bloodstream into solid tissue. In this study, we demonstrate that the blockade of lymphocyte extravasation potential mediates inhibition of certain cell-mediated immune responses in vivo. We investigated the effect of PT on the extravasation of antigen-primed lymphocytes into solid tissue. The results show that the loss of lymphocyte homing potential after in vitro treatment of the primed cells with PT is accompanied by an inhibition of antigen-specific contact hypersensitivity after adoptive transfer. This result suggests that the process of lymphocyte extravasation into nonlymphoid tissue sites such as the skin shares fundamental similarities to the selective localization of circulating lymphocytes to lymph nodes. Furthermore, the inhibition of contact hypersensitivity observed after the i.v. adoptive transfer of PT-treated antigen-primed cells could be circumvented by transferring the PT-treated cells directly into a tissue site with the relevant antigen. In contrast, no inhibition in the number of antibody-forming cells present within the spleen was observed after an adoptive transfer of PT-treated sheep red blood cell-primed lymphocytes, a result that is in agreement with radiotracer data. Thus, the inhibitory effect of PT on the adoptive transfer of contact hypersensitivity was established to be a direct result of the PT-mediated alteration of cellular migration, because PT inhibits the entrance of lymphocytes into specific tissue sites without inhibiting other cellular functions. This conclusion is additionally supported by experiments that showed that lymphocytes that have been pretreated with PT exhibit normal in vitro responses, as assayed by mitogenesis in response to concanavalin A and by the differentiation and function of alloantigen stimulated or hapten-specific cytotoxic T lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis

The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability.