Synthesis, surface properties, and biocompatibility of 1,2,3-triazole-containing alkyl ß-D-xylopyranoside surfactants.

We are interested in the development of surfactants derived from hemicellulosic biomass, as they are potential components in pharmaceuticals, personal care products, and other detergents. Such surfactants should exhibit low toxicity in mammalian cells. In this study we synthesized a series of alkyl or fluoroalkyl ß-xylopyranosides from azides and an alkyne using the copper-catalyzed azide-alkyne (CuAAC) 'click' reaction in 4 steps from xylose. The purified products were evaluated for both their surfactant properties, and for their biocompatibility. Unlike other carbohydrate-based surfactants, liquid-crystalline behavior was not observed by differential scanning calorimetry. The triazole-containing ß-xylopyranosides with short (6 carbons) and long (>12 carbons) chains exhibited no toxicity at concentrations ranging from 1 to 1000 µM. Triazole-containing ß-xylopyranosides with 8, 10, or 12 carbons caused toxicity via apoptosis, with CC50 values ranging from 26-890 µM. The two longest chain compounds did form stable monolayers at the air-water interface over a range of temperatures, although a brief transition to an the unstable monolayer was observed.

Disruption of phosphatidylcholine monolayers and bilayers by perfluorobutane sulfonate.

Perfluoroalkyl acids (PFAAs) are persistent environmental contaminants resistant to biological and chemical degradation due to the presence of carbon-fluorine bonds. These compounds exhibit developmental toxicity in vitro and in vivo. The mechanisms of toxicity may involve partitioning into lipid bilayers. We investigated the interaction between perfluorobutane sulfonate (PFBS), an emerging PFAA, and model phosphatidylcholine (PC) lipid assemblies (i.e., dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholine) using fluorescence anisotropy and Langmuir monolayer techniques. PFBS decreased the transition temperature and transition width of PC bilayers. The apparent membrane partition coefficients ranged from 4.9 × 10(2) to 8.2 × 10(2). The effects on each PC were comparable. The limiting molecular area of PC monolayers increased, and the surface pressure at collapse decreased in a concentration-dependent manner. The compressibility of all three PCs was decreased by PFBS. In summary, PFBS disrupted different model lipid assemblies, indicating potential for PFBS to be a human toxicant. However, the effects of PFBS are not as pronounced as those seen with longer chain PFAAs.

Synthesis, thermal properties, and cytotoxicity evaluation of hydrocarbon and fluorocarbon alkyl ß-D-xylopyranoside surfactants.

Alkyl ß-d-xylopyranosides are highly surface active, biodegradable surfactants that can be prepared from hemicelluloses and are of interest for use as pharmaceuticals, detergents, agrochemicals, and personal care products. To gain further insights into their structure-property and structure-activity relationships, the present study synthesized a series of hydrocarbon (-C(6)H(13) to -C(16)H(33)) and fluorocarbon (-(CH(2))(2)C(6)F(13)) alkyl ß-d-xylopyranosides in four steps from d-xylose by acylation or benzoylation, bromination, Koenigs-Knorr reaction, and hydrolysis, with the benzoyl protecting group giving better yields compared to the acyl group in the Koenigs-Knorr reaction. All alkyl ß-d-xylopyranosides formed thermotropic liquid crystals. The phase transition of the solid crystalline phase to a liquid crystalline phase increased linearly with the length of the hydrophobic tail. The clearing points were near constant for alkyl ß-d-xylopyranosides with a hydrophobic tail ⩾8, but occurred at a significantly lower temperature for hexyl ß-d-xylopyranoside. Short and long-chain alkyl ß-d-xylopyranosides displayed no cytotoxicity at concentration below their aqueous solubility limit. Hydrocarbon and fluorocarbon alkyl ß-d-xylopyranosides with intermediate chain length displayed some toxicity at millimolar concentrations due to apoptosis.

MAGIC Tool: integrated microarray data analysis.

SUMMARY: Several programs are now available for analyzing the large datasets arising from cDNA microarray experiments. Most programs are expensive commercial packages or require expensive third party software. Some are freely available to academic researchers, but are limited to one operating system. MicroArray Genome Imaging and Clustering Tool (MAGIC Tool) is an open source program that works on all major platforms, and takes users 'from tiff to gif'. Several unique features of MAGIC Tool are particularly useful for research and teaching. AVAILABILITY: http://www.bio.davidson.edu/MAGIC

Superoxide dismutase as a target for the selective killing of cancer cells.

Superoxide dismutases (SOD) are essential enzymes that eliminate superoxide radical (O2-) and thus protect cells from damage induced by free radicals. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Here we report that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes. Using complementary DNA microarray and biochemical approaches, we identify SOD as a target of this drug action and show that chemical modifications at the 2-carbon (2-OH, 2-OCH3) of the derivatives are essential for SOD inhibition and for apoptosis induction. Inhibition of SOD causes accumulation of cellular O2- and leads to free-radical-mediated damage to mitochondrial membranes, the release of cytochrome c from mitochondria and apoptosis of the cancer cells. Our results indicate that targeting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis.

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.

Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2).

We report the identification and characterization of a novel CC chemokine designated CCL28 and its receptor CCR10, known previously as orphan G-protein-coupled receptor GPR2. Human and mouse CCL28 share 83% identity at the amino acid and 76% at the nucleic acid levels. We also identified the mouse homologues of CCL28 and of CCR10, which map to mouse chromosomes 13 and 11, respectively. CCL28 is expressed in a variety of human and mouse tissues, and it appears to be predominantly produced by epithelial cells. Both human and mouse CCL28 induce calcium mobilization in human and mouse CCR10-expressing transfectants. CCL28 desensitized the calcium mobilization induced in CCR10 transfectants by CCL27, indicating that these chemokines share this new chemokine receptor. In vitro, recombinant human CCL28 displays chemotactic activity for resting CD4 or CD8 T cells.

Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC).

We recently reported the identification of a chemokine (CTACK), which has been renamed CCL27 according to a new systematic chemokine nomenclature. We report that CCL27 binds the previously orphan chemokine receptor GPR-2, as detected by calcium flux and chemotactic responses of GPR-2 transfectants. We renamed this receptor CCR10. Because of the skin-associated expression pattern of CCL27, we focused on the expression of CCL27 and CCR10 in normal skin compared with inflammatory and autoimmune skin diseases. CCL27 is constitutively produced by keratinocytes but can also be induced upon stimulation with TNF-alpha and IL-1beta. CCR10 is not expressed by keratinocytes and is instead expressed by melanocytes, dermal fibroblasts, and dermal microvascular endothelial cells. CCR10 was also detected in T cells as well as in skin-derived Langerhans cells. Taken together, these observations suggest a role for this novel ligand/receptor pair in both skin homeostasis as well as a potential role in inflammatory responses.

Identification of viral macrophage inflammatory protein (vMIP)-II as a ligand for GPR5/XCR1.

Lymphotactin is unique among chemokines in that it contains only two of four conserved cysteines and may possess a structure less constrained than other chemokines. The viral chemokine vMIP-II, which presumably has a structure similar to that of CC chemokines has been shown to inhibit many chemokine receptors, but its activity at GPR5/XCR1 has not been described. Interestingly, vMIP-II (but not vMIP-I) was found to be a potent antagonist of lymphotactin activity at GPR5/XCR1, extending the range of chemokine classes that this viral protein is known to inhibit to include the C class chemokine. In addition, we have extended previous analyses of GPR5/XCR1 expression and show that this receptor is expressed in leukocyte cells previously shown to be responsive to lymphotactin.

Comparison of action of paclitaxel and poly(L-glutamic acid)-paclitaxel conjugate in human breast cancer cells.

The new anticancer agent poly(L-glutamic acid)-paclitaxel (PG-TXL) is a conjugate of paclitaxel and the water-soluble polyglutamate carrier. The observation that PG-TXL appears to possess antitumor activity superior to free paclitaxel in preclinical studies suggests that PG-TXL might possess favorable pharmacokinetic properties and/or have a mechanism of action different from that of paclitaxel. The purpose of this study was to compare the pharmacological action of PG-TXL and free paclitaxel in a panel of breast cancer cell lines with emphasis on their ability to induce apoptosis, their effects on cell cycle progression, and their cellular uptake. Morphological analysis and biochemical characterizations demonstrated that both compounds have similar abilities to induce apoptosis in cells expressing wild-type p53 (MCF-7) or mutant p53 (MDA-MB435 and MDA-MB453). Although MCF-7 cells were less sensitive to each compound than MDA-MB435 and MDA-MB453 cells, transfection experiments demonstrated that p53 did not appear to play a significant role in drug-induced cell death with either agent. Flow cytometry analysis further revealed that both free paclitaxel and PG-TXL induced a characteristic G2/M arrest in the cell cycle, consistent with the disturbance of microtubule polymerization as their mechanism of action. Western blot analysis showed that paclitaxel and PG-TXL downregulated HER2/neu expression in a similar fashion. HPLC analysis revealed that paclitaxel was released from the PG-TXL conjugate in vitro. The released paclitaxel, not the glutamic acid polymer, was subsequently transported into the cells. These results suggest that PG-TXL exerts its anticancer activity by continuous release of free paclitaxel, and that the favorable pharmacokinetics and drug distribution of the PG-TXL conjugate in vivo are likely the main factors contributing to its superior anticancer activity.

CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells.

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA(+) memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA(+) T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.

The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells.

Dendritic cell migration to secondary lymphoid tissues is critical for Ag presentation to T cells necessary to elicit an immune response. Despite the importance of dendritic cell trafficking in immunity, at present little is understood about the mechanisms that underlie this phenomenon. Using a novel transwell chemotaxis assay system, we demonstrate that the CC chemokine receptor-7 (CCR7) ligands 6Ckine and macrophage inflammatory protein (MIP)-3 beta are selective chemoattractants for MHC class IIhigh B7-2high bone marrow-derived dendritic cells at a potency 1000-fold higher than their known activity on naive T cells. Furthermore, these chemokines stimulate the chemotaxis of freshly isolated lymph node dendritic cells, as well as the egress of skin dendritic cells ex vivo. Because these chemokines are expressed in lymphoid organs and 6Ckine has been localized to high endothelial venules and lymphatic endothelium, we propose that they may play an important role in the homing of dendritic cells to lymphoid tissues.

Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites.

DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34(+) hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-alpha or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34(+) HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3alpha, but also to MIP-1alpha and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-alpha, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3beta. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3alpha and MIP-3beta, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3beta responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3beta is also acquired by CD11c+ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3alpha mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3beta mRNA specifically in T cell-rich areas, suggests a role for MIP-3alpha/CCR6 in recruitment of immature DCs at site of injury and for MIP-3beta/CCR7 in accumulation of antigen-loaded mature DCs in T cell-rich areas.

Molecular cloning and functional characterization of human MIP-1 delta, a new C-C chemokine related to mouse CCF-18 and C10.

We have isolated a novel human C-C chemokine, MIP-1 delta from a human fetal spleen cDNA library. The human MIP-1 delta cDNA has an unusually long 400-bp 5-prime untranslated region and a predicted 113-amino acid protein of 10 kDa. The coding sequence contains a signal peptide of 21 amino acids, indicating that the mature protein has 92 amino acids (8 kDa). Recombinant human MIP-1 delta produced by transfected human embryonic kidney 293 cells produced an 8-kDa protein, which confirmed the presence of a signal peptide. Compared with other human C-C chemokines, human MIP-1 delta shows the highest homology with human HCC-1, CK beta-8, murine C10, and CCF18 (MIP-1 gamma). The human MIP-1 delta gene is localized on chromosome 17 where most of the C-C chemokine superfamily is located. Human MIP-1 delta is expressed in T and B lymphocytes, NK cells, monocytes, and monocyte-derived dendritic cells, but not in bone marrow-derived dendritic cells. Its expression can be induced by other proinflammatory cytokines in monocytes and dendritic cells. Human MIP-1 delta is chemotactic for T cells and monocytes, but not for neutrophils, eosinophils, or B cells. Human MIP-1 delta induced calcium flux in human CCR1-transfected cells.

RANTES-induced T cell activation correlates with CD3 expression.

The chemokine RANTES induces a unique biphasic cytoplasmic Ca2+ signal in T cells. The first phase of this signal, similar to that of other chemokines, is G-protein mediated and chemotaxis associated. The second phase of this signal, unique to RANTES and evident at concentrations greater than 100 nM, is tyrosine kinase linked and results in a spectrum of responses similar to those seen with antigenic stimulation of T cells. We show here that certain jurkat T cells responded to RANTES solely through this latter pathway. A direct correlation between the RANTES-induced second phase response and CD3 expression was demonstrated in these cells. Sorting the Jurkat cells into CD3(high) and CD3(low) populations revealed that only the CD3(high) cells were responsive to RANTES. Furthermore, stimulation of these Jurkat cells with anti-CD3 mAb significantly depresses their subsequent response to RANTES. While a RANTES-specific chemokine receptor is expressed at a low level on these Jurkat cells, the RANTES-induced activation is dependent on the presence of the TCR. Thus, stimulation through TCR may partially account for RANTES' unique pattern of signaling in T cells.

Chemokine receptor CCR3 function is highly dependent on local pH and ionic strength.

The CC chemokine receptor 3 (CCR3) plays an important role in the regulation of the migration of eosinophils, a leukocyte population involved in many inflammatory pathologies including asthma. CCR3 binds to the CC chemokine eotaxin, a promigratory cytokine originally isolated as the key component in a model of eosinophil-induced airway inflammation. We show here that eotaxin/CCR3 binding interactions exhibit a marked sensitivity to relatively small changes in the extracellular environment. In particular, modest variations in the pH and the level of sodium chloride over a range of physiologic and near physiologic conditions had dramatic effects on eotaxin binding and CCR3-mediated cytoplasmic Ca2+ mobilization. These biochemical indices were reflected at the functional level as well; small changes in pH and salt also resulted in striking changes in the migration of primary human eosinophils in vitro. These results reveal that relatively small perturbations in extracellular buffer conditions can yield widely disparate interpretations of CCR3 ligand binding and affinities and suggest that modulation of the tissue microenvironment might be utilized to control the affinity and efficacy of chemokine-mediated cell migration.

Characterization of transgenic pigs expressing functionally active human CD59 on cardiac endothelium.

The critical shortage of human donor organs has generated interest in the potential for porcine to human xenotransplantation. The initial immunological barrier to xenotransplantation is hyperacute rejection, which is mediated by xenoreactive antibodies and complement, and results in rapid and irreversible tissue destruction. While endogenous complement regulatory proteins (CRPs) protect cells from injury caused by autologous complement, they are relatively species specific and most likely ineffectual in this setting. This has led to the hypothesis that expression of human CRPs in transgenic pigs may affect susceptibility to complement-mediated tissue injury in a porcine-to-human xenograft. Using specific lines of transgenic pigs that express low levels of human CD59, a CRP that acts at the terminal stage of the complement cascade, we present evidence that shows that the human CD59 protein inhibits membrane attack complex assembly and reduces tissue damage when the heart is transplanted to a baboon. Examination by immunohistochemistry of transgenic porcine hearts after transplantation revealed markedly reduced deposition of C5b and MAC, but a similar level of C3 deposition as compared with transplanted control hearts. This finding supports the concept that the species specific function of CRPs contributes to the humoral barrier to xenotransplantation and, given the low level of human CD59 protein expression in the porcine heart, argues that the human protein contributes a unique rather than an additive function in regulation of complement in a xenogeneic setting.

Human CD59 expressed in transgenic mouse hearts inhibits the activation of complement.

Porcine-to-human xenotransplantation offers a potential solution to the critical shortage of human organs. The major immunological barrier to xenotransplantation between these species is a rapid rejection process mediated by preformed natural antibodies and complement. Xenogeneic organ grafts are especially susceptible to complement mediated injury because complement regulatory proteins, which ordinarily protect cells from inadvertent injury during the activation of complement, function poorly in regulating activation of heterologous complement. Removal of xenoreactive antibodies or systemic inhibition of complement activity has been shown to prolong graft survival. As an alternative to the systemic inhibition of complement activity, we have established a model system using transgenic animals to test whether the expression of human membrane bound complement regulatory proteins on mouse endothelial cells can inhibit the activation of human complement. CD59, which acts at the terminal stage of complement activation by inhibiting the formation of the membrane attack complex, was used as a paradigm for this model. A CD59 construct containing the putative CD59 gene promoter linked to the CD59 coding region was used to demonstrate expression of the human CD59 protein in various tissues of transgenic mice, including endothelial cells in the heart. In addition, we show that the transgenic CD59 protein is biologically active as determined by the ability to inhibit the formation of membrane attack complex in transgenic mouse hearts perfused ex vivo with human plasma. These results demonstrate that expression of membrane bound complement regulatory proteins can achieve complement inhibition in a xenogeneic organ and suggest that this approach may be useful for successful xenotransplantation between discordant species.

Regulation of growth hormone receptor and binding protein expression in domestic species.

Growth hormone receptor (GHR) expression has been analyzed at the RNA level. In the rat, relative expression of the RNA species encoding the GHR and the GH-binding protein (GHBP) appears to be sensitive to endocrine status. Full-length GHR cDNA clones from ovine, porcine, and chicken were used as probes to investigate the existence of unique RNAs for GHBPs in these species. In the sheep and pig, only a single, approximately 4.5-kb RNA is apparent. Although quite high levels of GH binding activity are found in pig serum, a variety of methods failed to isolate a separate GHBP message, suggesting that porcine GHBP is produced via a mechanism different from that which is known for rat. One class of chicken GHR cDNA, resulting from alternative use of a splice acceptor 17 bases upstream of the intron 6/Exon 7 junction, is also presented.