HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.     

Soybean aeroallergen around the port of New Orleans: A potential cause of asthma

There have been reported epidemics of severe asthma in Barcelona, Spain, linked to a 10 kDa low molecular mass (LMM) allergen from soybean hulls that became airborne during unloading of ships. As a preliminary probe of the potential for dispersion of this allergen in USA cities, four automated air samplers were placed around a grain elevator in New Orleans and operated continuously from May to October 1990. The allergen was extracted from the filters and immunochemically assayed for soybean aeroallergen. On 31 separate days, the airborne allergen concentration in at least one of the samples was over 10000 U/m³ similar to those observed in Barcelona on some epidemic days. Areas North and East of the elevator were most affected. Serologie studies showed that of 50 asthmatics from New Orleans who were participants in an unrelated clinical study 4 or 8% demonstrated elevated titers of IgE antibody to LMM soybean allergen. Only 1 of 475 control sera (half of which were also asthmatic) obtained elsewhere in the US was positive for LMM soybean IgE antibody. Based on the findings in this study, there is a great possibility that on some days there is enough soybean allergen in the air and a sufficient frequency of soybean aeroallergen RAST positive asthmatics in New Orleans to warrant further investigation of the contribution of soybean aeroallergen to asthma around the port of New Orleans.Supported by NIAID # A121255. Mayo Clinic and Foundation and Minnesota Lung Association.     

Myosin binding protein C interaction with actin: characterization and mapping of the binding site

Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and shown that purified cMyBPC binds actin filaments with an affinity of 4.3 ± 1.1 μM and a 1:1 molar ratio with regard to an actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. To determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of the cMyBPC sequence. Recombinant N-terminal fragments such as C0-C1, C0-C4, and C0-C5 cosediment with actin in a linear, nonsaturable manner. At the same time, MyBPC fragments lacking either the C0-C1 or C0-C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via nonspecific electrostatic interactions.     

N-glycosylation modulates the cell-surface expression and catalytic activity of corin

N-Glycosylation may influence the subcellular localization and biological activity of the pro-ANP convertase, corin. In HEK293-corin cells, the inhibition of N-glycosylation, with tunicamycin, reduced the cell-surface expression of murine corin, but did not alter the total expression. Therefore, tunicamycin treatment likely caused the intracellular accumulation of non-glycosylated corin. Tunicamycin treatment also significantly reduced corin activity (pro-ANP cleavage) in these cells. We developed an assay to measure the effect of N-glycosylation on corin activity, independent of its effect on corin localization. We determined that the reduction in corin activity was due to a direct effect of N-glycosylation, and was not secondary to the effect of N-glycosylation on corin cell-surface expression. Our data provide evidence that N-glycosylation is essential for the cell-surface expression of murine corin and modulates its functional activity. N-Glycosylation represents a possible mechanism for the regulation of native corin on the surface of cardiomyocytes.     

The tomato early fruit specific gene <Emphasis Type="Italic">Lefsm1</Emphasis> defines a novel class of plant-specific SANT/MYB domain proteins

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.     

Pivotal Role of Mitochondrial Calcium Uptake in Neural Cell Apoptosis and Necrosis

Abstract : Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca²⁺]C) and intramitochondrial ([Ca²⁺]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine ; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal ; HNE) insult. STS and HNE each induced an early increase of [Ca²⁺]C followed by delayed increase of [Ca²⁺]M. Overexpression of Bcl-2 blocked the elevation of [Ca²⁺]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca²⁺]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca²⁺]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.