Characterization of human CD4 helper T cell responses against Aurora kinase A

Aurora kinase A (Aurora-A) is a cell cycle-associated serine–threonine kinase that is overexpressed by various types of cancer and is highly associated with poor prognosis. Since the expression of Aurora-A in normal tissues has been shown to be significantly lower as compared to tumor cells, this protein is being considered as a potential tumor-associated antigen for developing immunotherapies. The goal in the present study was to identify CD4 helper T lymphocyte (HTL) epitopes for Aurora-A for the design of T cell-based immunotherapies against Aurora-A-expressing tumors. Synthetic peptides corresponding to potential HTL epitopes were identified from Aurora-A and used to stimulate CD4 T lymphocytes in vitro to generate antigen-specific HTL clones that were evaluated for antigen specificity, MHC restriction and for their ability to interact with Aurora-A-expressing tumor cells. The results show that two peptides (Aurora-A_161–175 and Aurora-A_233–247) were effective in generating HTL responses that were restricted by more than one MHC class II allele (i.e., promiscuous responses). The CD4 HTL clones were able to directly recognize Aurora-A-expressing tumor cells in an antigen-specific and MHC class II-restricted manner and some of the clones displayed cytolytic activity toward Aurora-A + tumor cells. Both of these peptides were capable of stimulating in vitro T cell responses in patients with bladder cancer.     

Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine,N,N′-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N′-diacetylchitobiose [(GlcNAc)₂] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.     

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.     

Loss of stearoyl-CoA desaturase 1 rescues cardiac function in obese leptin-deficient mice

The heart of leptin-deficient ob/ob mice is characterized by pathologic left ventricular hypertrophy along with elevated triglyceride (TG) content, increased stearoyl-CoA desaturase (SCD) activity, and increased myocyte apoptosis. In the present study, using an ob/ob;SCD1^−/− mouse model, we tested the hypothesis that lack of SCD1 could improve steatosis and left ventricle (LV) function in leptin deficiency. We show that disruption of the SCD1 gene improves cardiac function in ob/ob mice by correcting systolic and diastolic dysfunction without affecting levels of plasma TG and FFA. The improvement is associated with reduced expression of genes involved in FA transport and lipid synthesis in the heart, as well as reduction in cardiac FFA, diacylglycerol, TG, and ceramide levels. The rate of FA β-oxidation is also significantly lower in the heart of ob/ob;SCD1^−/− mice compared with ob/ob controls. Moreover, SCD1 deficiency reduces cardiac apoptosis in ob/ob mice due to increased expression of antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase and caspase-3 activities. Reduction in myocardial lipid accumulation and inhibition of apoptosis appear to be one of the main mechanisms responsible for improved LV function in ob/ob mice caused by SCD1 deficiency.     

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis—the ApoE-deficient mouse—by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet–fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.     

Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca2+-independent phospholipase A2γ-deficient mice

Group VIB Ca²⁺-independent phospholipase A₂γ (iPLA₂γ) is a membrane-bound iPLA₂ enzyme with unique features, such as the utilization of distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. Here we investigated the physiological functions of iPLA₂γ by disrupting its gene in mice. iPLA₂γ-knockout (KO) mice were born with an expected Mendelian ratio and appeared normal and healthy at the age of one month but began to show growth retardation from the age of two months as well as kyphosis and significant muscle weakness at the age of four months. Electron microscopy revealed swelling and reduced numbers of mitochondria and atrophy of myofilaments in iPLA₂γ-KO skeletal muscles. Increased lipid peroxidation and the induction of several oxidative stress-related genes were also found in the iPLA₂γ-KO muscles. These results provide evidence that impairment of iPLA₂γ causes mitochondrial dysfunction and increased oxidative stress, leading to the loss of skeletal muscle structure and function. We further found that the compositions of cardiolipin and other phospholipid subclasses were altered and that the levels of myoprotective prostanoids were reduced in iPLA₂γ-KO skeletal muscle. Thus, in addition to maintenance of homeostasis of the mitochondrial membrane, iPLA₂γ may contribute to modulation of lipid mediator production in vivo.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.