Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M r > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M r 230,000) and CD47 (apparent M r > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser 64 and Ser 79 . Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser 64 .Cell surface proteoglycans play critical roles in cell-matrix interactions. They act as co-receptors for some pathogens and growth and motility factors and regulate the functions of other cell surface signaling receptors (reviewed in Refs. 1-4). Chondroitin sulfate or heparan sulfate chains are attached through a conserved core oligosaccharide sequence to specific Ser residues on the core proteins (5). The most prevalent cell surface heparan sulfate proteoglycans (HSPG) 5 are the syndecans and glypicans, but a number of additional cell surface proteins can be specifically modified with heparan sulfate glycosaminoglycans (GAG) in certain cells and tissues (6). CD4 ϩ T cells express highly sulfated heparan sulfate chains because of their high expression of the N-deacetylase/N-sulfotransferase NDST2 and the 3-O-sulfotransferases 3-OST3 (7). T cell HSPGs have been identified as receptors for histones (8) and cyclophilin B (9, 10) and serve as co-receptors for heparinbinding chemokines such as regulated on activation normal T cell expressed and secreted (RANTES) (11).T cell HSPGs are also co-receptors for retroviral infection. An unidentified cell surface HSPG expressed by the H9 T cell line interacts with the V3 region of the HIV1 envelope gp120-gp41, and heparitinase treatment inhibited binding and entry of the virus into H9 cells (12). The fusion peptide domain of gp41 also interacts specifically with an HSPG on T cells (13). A subsequent study concluded that syndecan functions in trans to promote HIV infection of T cells (14). Similar HSPG binding was demonstrated for the hu...

We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We showed that recombinant full-length human TSG-6 (TSG-6Q) and the Link module of TSG-6 (Link_TSG6) bind 125 I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin results from its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca 2؉ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation dependence is due to conformational effects of calcium-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of the inter-␣-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.

Human plasma fibronectin binds with high affinity to the inflammation-induced secreted protein TSG-6. Fibronectin binds to the CUB_C domain of TSG-6 but not to its Link module. TSG-6 can thus act as a bridging molecule to facilitate fibronectin association with the TSG-6 Link module ligand thrombospondin-1. Fibronectin binding to TSG-6 is divalent cation-independent and is conserved in cellular fibronectins. Based on competition binding studies using recombinant and proteolytic fragments of fibronectin, TSG-6 binding localizes to type III repeats 9-14 of fibronectin. This region of fibronectin contains the Arg-Gly-Asp sequence recognized by α5β1 integrin, but deletion of that sequence does not prevent TSG-6 binding, and TSG-6 does not inhibit cell adhesion on fibronectin substrates mediated by this integrin. This region of fibronectin is also involved in fibronectin matrix assembly, and addition of TSG-6 enhances exogenous and endogenous fibronectin matrix assembly by human fibroblasts. Therefore, TSG-6 is a high affinity ligand that can mediate fibronectin interactions with other matrix components and modulate some interactions of fibronectin with cells.

We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFα-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.

In recent years, new prospects for the use of nucleic acids as anticancer drugs have been discovered. Aptamers for intracellular targets can regulate cellular functions and cause cell death or proliferation. However, intracellular aptamers have limited use for therapeutic applications due to their low bioavailability. In this work, we selected DNA aptamers to cell organelles and nucleus of cancer cells, and showed that an aptamer NAS-24 binds to vimentin and causes apoptosis of mouse ascites adenocarcinoma cells in vitro and in vivo. To deliver the aptamer NAS-24 inside cells, natural polysaccharide arabinogalactan was used as a carrier reagent. The mixture of arabinogalactan and NAS-24 was injected intraperitonealy for 5 days into mice with adenocarcinoma and inhibited adenocarcinoma growth more effectively than free arabinogalactan or the aptamer alone. The use of aptamers to intracellular targets together with arabinogalactan becomes a promising approach for anticancer therapy.

Disseminated Candida albicans infection results in high morbidity and mortality despite treatment with existing antifungal drugs. Recent studies suggest that modulating the host immune response can improve survival, but specific host targets for accomplishing this goal remain to be identified. The extracellular matrix protein thrombospondin-1 is released at sites of tissue injury and modulates several immune functions, but its role in C. albicans pathogenesis has not been investigated. Here, we show that mice lacking thrombospondin-1 have an advantage in surviving disseminated candidiasis and more efficiently clear the initial colonization from kidneys despite exhibiting fewer infiltrating leukocytes. By examining local and systemic cytokine responses to C. albicans and other standard inflammatory stimuli, we identify a crucial function of phagocytes in this enhanced resistance. Subcutaneous air pouch and systemic candidiasis models demonstrated that endogenous thrombospondin-1 enhances the early innate immune response against C. albicans and promotes activation of inflammatory macrophages (inducible nitric oxide synthase+, IL-6high, TNF-αhigh, IL-10low), release of the chemokines MIP-2, JE, MIP-1α, and RANTES, and CXCR2-driven polymorphonuclear leukocytes recruitment. However, thrombospondin-1 inhibited the phagocytic capacity of inflammatory leukocytes in vivo and in vitro, resulting in increased fungal burden in the kidney and increased mortality in wild type mice. Thus, thrombospondin-1 enhances the pathogenesis of disseminated candidiasis by creating an imbalance in the host immune response that ultimately leads to reduced phagocytic function, impaired fungal clearance, and increased mortality. Conversely, inhibitors of thrombospondin-1 may be useful drugs to improve patient recovery from disseminated candidiasis.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chimera (TFO-P) can specifically recognise its DNA target at physiological salt and pH conditions. Bound to the double-stranded target DNA in a promoter region, the TFO-P is able to activate gene expression. Our results suggest that this type of molecule may prove useful in the design of new tools for artificial modulation of gene expression.

Two ways of catalytic depolymerization of native and isolated wood lignins are described: the peroxide delignification of hardwood (aspen, birch) and softwood (abies) in the medium of acetic acidwater over TiO 2 catalyst and the thermal dissolution of organosolv lignins (ethanol-lignin and acetone-lignin) in supercritical alcohols (ethanol and butanol) over solid Ni-containing catalysts. The catalyst TiO 2 in rutile modification has the higher activity in wood peroxide delignification at 100°C as compared to TiO 2 in anatase modification. The results of kinetic studies and optimization of the processes of peroxide depolymerization of hardwood (aspen, birch) and softwood (abies) lignins in the medium of acetic acidwater over catalyst TiO 2 (rutile) at mild conditions (≤ 100°C, atmospheric pressure) are compared. The catalyst TiO 2 initiates the formation of OH • and OOH • radicals from H 2 O 2 which promote the oxidative fragmentation of wood lignin. In this case, the peroxide depolymerization of softwood lignin, constructed from phenylpropane units of guaiacyl-type proceeds more difficult than the hardwood lignins, mainly containing syringyl-type units. The solid and soluble products of peroxide catalytic delignification of wood under the optimized conditions were studied by FTIR, XRD, GC-MS and chemical methods. Regardless of the nature of wood the cellulosic products have a structure similar to microcrystalline cellulose. The soluble products mainly consist of monosaccharides and organic acids. Aromatic compounds are present only in a low amount which indicates the oxidative degradation of aromatic rings of lignin phenylpropane units under the used conditions of wood catalytic delignification. The processes of thermal dissolution of acetone-lignin and ethanol-lignin from aspenwood in supercritical ethanol and butanol over Ni-containing catalyst (NiCu/SiO 2 , NiCuMo/SiO 2) are compared. The composition, structure and thermal properties of organosolv lignins were studied with the use of FTIR, GPC, 1 H-13 C HSQC NMR, DTA and elemental analysis. The influence of a composition of Ni-containing catalyst on the thermal conversion in supercritical butanol and ethanol of ethanol-lignin and acetone-lignin was established. The highest conversion of lignins (to 93% wt.) in supercritical alcohols and the highest yield of liquid products (to 90 % wt.) were achieved at 300 °C in the presence of catalyst NiCuMo/SiO 2. Scheme of green biorefinery of wood based on the use of non-toxic and low-toxic reagents (H 2 O 2 , H 2 O, acetic acid, ethanol, butanol) and solid catalysts (TiO 2 , NiCuMo/SiO 2) is suggested.