A metalloproteinase inhibitor blocks the shedding of soluble cytokine receptors and processing of transmembrane cytokine precursors in human monocytic cells

A number of membrane-anchored cytokines and cytokine receptors are susceptible to yield soluble counterparts. Recently, peptide-hydroxamate metalloproteinase inhibitors have been reported to block the proteolytic processing of tumour necrosis factor (TNF)-alpha 55- and 75-kDa TNF receptors (TNF-R55 and TNF-R75), and interleukin (IL)-6R. In this report the authors studied the effect of an hydroxamate metalloproteinase inhibitor on the secretion of cytokines and the generation of cytokine soluble receptors by human myelomonoycytic cell lines and purified monocytes. Whereas secretion of cytokines lacking a transmembrane domain precursor (IL-1 alpha, IL-1 beta, IL-6 or IL-10) is either unaffected or augmented, shedding/secretion of transmembrane domain-containing cytokines and cytokine receptors [TNF-alpha, macrophage colony-stimulating factor (M-CSF), transforming growth factor (TGF)-alpha, stem cell factor (SCF), TNF-R55, TNF-R75, and IL-6R] was dramatically decreased in the presence of the metalloproteinase inhibitor. The diversity of sequences in the cleavage site of these proteins and differences found in the inhibitory concentration values suggest the existence of a metalloproteinase family displaying different substrate specificity. These results emphasize the important role of metalloproteinases as regulators of membrane expression and secretion of cytokines and cytokine receptors.     

Tyrosine phosphorylation of transmembrane ligands for Eph receptors

Axonal pathfinding in the nervous system is mediated in part by cell-to-cell signaling events involving members of the Eph receptor tyrosine kinase (RTK) family and their membrane-bound ligands. Genetic evidence suggests that transmembrane ligands may transduce signals in the developing embryo. The cytoplasmic domain of the transmembrane ligand Lerk2 became phosphorylated on tyrosine residues after contact with the Nuk/Cek5 receptor ectodomain, which suggests that Lerk2 has receptorlike intrinsic signaling potential. Moreover, Lerk2 is an in vivo substrate for the platelet-derived growth factor receptor, which suggests crosstalk between Lerk2 signaling and signaling cascades activated by tyrosine kinases. It is proposed that transmembrane ligands of Eph receptors act not only as conventional RTK ligands but also as receptorlike signaling molecules.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

Elk-L3, a novel transmembrane ligand for the Eph family of receptor tyrosine kinases, expressed in embryonic floor plate, roof plate and hindbrain segments

The Eph family of receptor tyrosine kinases has 13 distinct members and seven ligands for these receptors have been described to date. These receptors and their ligands have been implicated in regulating neuronal axon guidance and in patterning of the developing nervous system and may also serve a patterning and compartmentalization role outside of the nervous system as well. The ligands are all membrane-attached, and this attachment appears to be crucial for their normal function; five of the known ligands are linked to the membrane via a glycosyl phosphotidylinositol (GPI) linkage, while two of the ligands are transmembrane proteins. Despite the large number of Eph family receptors and ligands, they can be divided into just two major subclasses based on their binding specificities. All the GPI-anchored ligands bind and activate one subclass of the Eph receptors (that represented by Eck) while the two transmembrane ligands bind and activate the other major subclass of receptors (represented by Elk). Here we report the identification and characterization of the third, and most divergent, member of the transmembrane group of Eph ligands, which we term Elk-L3 (Elk-related receptor ligand number 3). Elk-L3 is notable for its remarkably restricted and prominent expression in the floor plate and roof plate of the developing neural tube and its rhombomere-specific expression in the developing hindbrain. The Elk-L3 gene has been localized to mouse chromosome 11 and human chromosome 17.     

Intracochlear factors contributing to psychophysical percepts following cochlear implantation

Glycosyl-phosphatidylinositol (GPI) lipids have a structural role as protein anchors to the cell surface. In addition, they are implicated in hormone, growth factor and cytokine signal transduction. Their phosphodiesteric hydrolysis mediated by an activated phospholipase results in the generation of water soluble oligosaccharide species termed the inositol phosphoglycan (IPG). This product has been demonstrated to possess biological properties when added exogenously to cells mimicking the biological effects of a variety of extracellular ligands. This may be accomplished since IPG is generic for a family of closely related species which are released in a tissue-specific manner and additionally have cell-specific targets. Micro-organic synthesis has recently been able to shed new light on this topic by the introduction of defined oligosaccharide analogues of IPG for the assessment of their biological activity. These have complemented the findings observed with purified IPG from biological sources thus strengthening the belief that the GPI/IPG signalling system represents a truly novel aspect of transmembrane signalling.     

Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells

BACKGROUND/AIMS: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-alpha and interferon-gamma on proliferation, "activation" and protein synthesis of hepatic stellate cells. METHODS: Primary cultures of hepatic stellate cells were exposed to tumour necrosis factor-alpha and interferon-gamma. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western- and Northern blotting techniques. RESULTS: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-alpha and interferon-gamma, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle alpha-actin and of TGF-beta/activin type I receptor was induced by tumour necrosis factor-alpha but downregulated by interferon-gamma. Tumour necrosis factor-alpha increased the synthesis of distinct extracellular matrix proteins, particularly of fibronectin and tenascin, but decreased collagen type III expression. In contrast, interferon-gamma reduced the synthesis of all connective tissue proteins tested. Among the protease inhibitors, interferon-gamma induced C1-esterase inhibitor synthesis, while tumour necrosis factor-alpha stimulated plasminogen activator inhibitor type 1 production. CONCLUSIONS: Tumour necrosis factor-alpha and interferon-gamma decrease proliferation of hepatic stellate cells, while "activation" of hepatic stellate cells and synthesis of proteins involved in matrix metabolism are regulated in a differential, cytokine-specific manner, suggesting that both cytokines play an important role in liver repair.     

Structure of a heparin-linked biologically active dimer of fibroblast growth factor

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.     

Screening lab tests of Chlamydia trachomatis. Do they show declining trend of the infections?

Peptides are flexible molecules and can adopt local structural features of protein, such as secondary structure, hydrophobicity, and distribution of electrostatic charges, and so forth, and mimic their functions. Therapeutic peptidomimetics that are immunologically relevant are developed by engineering the surface loop structures in the proteins and receptors. The class of molecules targeted include immunoglobulin fold-containing molecules: antibody, cell-surface CD4 receptors and cystine-knot-containing receptor family members: tumor necrosis factor (TNF), CD40, and p185/Neu receptors. We have used the loops involved in the molecular recognition as a template and developed peptidomimetics that interfere with the functions of the target molecules. In this article, two molecular targets are discussed: (1) immunoglobulin fold-containing CD4 receptor and (2) cystine-knot-containing TNF receptor (TNFR).     

TIMP-3 induces cell death by stabilizing TNF-alpha receptors on the surface of human colon carcinoma cells

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) regulate the structural integrity of the extracellular matrix (ECM). Constitutive expression of human TIMP-3 in human DLD colon carcinoma cells renewed serum-responses and inhibited tumour formation in nude mice. To elucidate the mechanism of TIMP-3-mediated tumour suppression, we compared parental DLD and TIMP-3 expressing DLD cells (TIMP-3/DLD), finding them to be significantly different. TIMP-3/DLD cultures have fewer mitotic cells, are delayed in G1, and die after serum starvation. TIMP-3/DLD conditioned media activates cell death on fibroblast cells. The cell death induced by serum starvation and conditioned media was inhibited by 70%, in the presence of neutralizing tumour necrosis factor alpha (TNF-alpha) antibody. TIMP-3/DLD whole cell lysate contained p55 TNF-alpha receptor, while vector/DLD lysate had p55 TNF-alpha receptor and p46 soluble TNF-alpha inhibitor. Vector/DLD conditioned media had p46, while no soluble TNF-alpha receptor was detected in TIMP-3/DLD conditioned media. In addition, FACS analysis revealed that TIMP-3/DLD cells have more TNF-alpha surface binding sites, suggesting a direct correlation between TIMP-3 expression and surface receptors. The mechanism of tumorigenic reversion induced by TIMP-3 in DLD cells may involve protection of receptors from the proteolytic activity of MMPs. Putative TIMP-3-mediated inhibition of MMPs restores the TNF-alpha p55 signalling pathway and the carcinoma cell is killed by autocrine TNF-alpha. Thus, DLD cells have specific ECM MMPs that cleave cytokines and cytokine receptors. TIMP-3 specifically inhibits MMPs involved in receptor shedding.     

Molecular aspects of pheromonal communication via the vomeronasal organ of mammals

Recently, two large multigene families of putative G-protein-linked receptors that are expressed in distinct subpopulations of neurones in the vomeronasal organ have been identified. These receptors probably mediate pheromone detection. The most surprising aspects of these findings are that there are so many receptors of two very different classes and that the receptors are unrelated to their counterparts in the main olfactory epithelium. This suggests that many active ligands are likely to exert effects through the vomeronasal organ. Parallel experiments addressing the nature of these ligands indicate a role for some proteins, as well as small molecules, as functional mammalian pheromones. In combination, these results begin to suggest a molecular basis for mammalian pheromone signalling.     

Pharmacokinetics of fusidic acid after a single dose of a new paediatric suspension

Cell proliferation in response to growth factors is mediated by specific high affinity receptors. Ligand-binding by receptors of the protein tyrosine kinase family results in the stimulation of several intracellular signal transduction pathways. Key signalling enzymes are recruited to the plasma membrane through the formation of stable complexes with activated receptors. These interactions are mediated by the conserved, non-catalytic SH2 domains present in the signalling molecules, which bind with high affinity and specificity to tyrosine-phosphorylated sequences on the receptors. The assembly of enzyme complexes is emerging as a major mechanism of signal transduction and may regulate the pleiotropic effects of growth factors.     

Genomic organization and allelic polymorphism of the human killer cell inhibitory receptor gene KIR103

Killer cell inhibitory receptors (KIR) belong to the immunoglobulin superfamily of molecules and are expressed on natural killer (NK) cells. The KIRs of the p58/p50 family have two immunoglobulin domains and are ligands for HLA-Cw antigens, whereas the p70/p70 delta family has three immunoglobulin domains and comprises ligands for HLA-B antigens and possibly some HLA-A antigens. Members of a third KIR family, KIR103, have two immunoglobulin domains but have highest nucleotide sequence homology to the p70 family. The ligands for KIR103 on target cells are currently unknown. We here report the complete genomic organization of KIR103. It spans about 12 kb of DNA and consists of eight exons of which exon 1 and exon 2 encode the leader sequence. Exon 3 encodes the first immunoglobulin domain (gamma 1), and exon 4 encodes the main part of the second immunoglobulin domain (gamma 3), which also contains sequences contributed by exon 5 and exon 6. Exon 6 encodes the transmembrane domain, whereas exons 7 and 8 encode most of the cytoplasmic domain. KIR103 is polymorphic, and two alleles, 103AS and 103LP, are defined in this study. Additional full-length cDNA clones for KIR103 have been isolated and are shown to be formed by alternative mRNA splicing with exon skipping. Some of these truncated KIR103 cDNA could encode shorter transmembrane molecules, whereas others lack the transmembrane domain and are candidate genes for secreted KIR products. KIR103 is localized to the KIR genetic region on chromosome 19q13.4.     

Subjective evaluation of profile prediction using video imaging

Several cytokines and growth factors together with their binding proteins and/or receptors are being increasingly detected in mammalian thyroid tissue. These include epidermal growth factor, insulin-like growth factors, transforming growth factors, fibroblast growth factor, interleukins, interferons, and tumour necrosis factor-alpha. Their exact role in relation to thyroid regulation has not been fully elucidated but it is clear that many of these peptide regulatory factors are mitogenic for cultured thyrocytes. Recent evidence suggests that some thyroid cancers and benign goiters over-express receptors for a number of these growth factors. Transforming growth factor-beta is unique for its dominant anti-proliferative effect on thyrocytes concurrently exposed to potent thyroid mitogens. The mammalian thyroid cell is clearly a source as well as target of myriad polypeptide factors that probably co-regulate its normal growth and differentiation. Aberrant expression of these growth factors or their receptors might be a factor in the development of goiter.     

The Fas antigen and Fas-mediated apoptosis in B-cell differentiation

In the B-cell lineage, Fas, a type 1 membrane protein belonging to the tumor necrosis factor receptor (TNF) family, is expressed on B-cells at a restricted developmental stage and on activated B-cells, but not on naive mature B-cells. Apoptosis mediated by Fas-Fas ligand interactions may be involved in the peripheral elimination of autoreactive B-cells and in the regulation of the immune response through deletion of B-cells activated by foreign antigens, as for the T-cell lineage. Fas-mediated apoptosis associated with B-cell activation is affected by costimulation through other accessory signaling molecules like CD40, whose ligands are on T-cells.     

Effect of cardiolipin on functional properties of isolated rat liver mitochondria

Receptor-type serine/threonine kinases (RSKs) have been organized into two distinct classes known as types I and II on the basis of sequence similarity. However, experiments have shown ligand specificities in the two classes and as a result type I and type II receptors can often bind to a common ligand. The transforming growth factor-beta- (TGF-beta) specific receptors represent such a case, where both type I and II receptors (T beta RI and T beta RII) are observed. Of additional interest is the observation that heteromeric associations of type I and II receptors can also enable signaling. To further elucidate the function of various RSKs, the extracellular domains of both alpha and beta chains from human granulocyte-macrophage colony-stimulating factor receptors were linked to transmembrane cytoplasmic domains of RSKs. Chimeric receptors of human granulocyte-macrophage receptor (hGMR) alpha with T beta RI and hGMR beta with T beta RII were expressed in murine pre-B cell-derived Ba/F3 cells. These chimeras formed heteromeric complexes, transmitted TGF-beta signals, and were down-modulated in response to human granulocyte-macrophage colony-stimulating factor. However, experiments utilizing these chimeric receptors in different combinations revealed that only heteromeric associations of transmembrane cytoplasmic domains mediated signaling and down-modulation. Chimeric receptors with transmembrane cytoplasmic domains of activin receptor type II and bone morphogenetic protein receptor type II also provided signals in conjunction with chimeric T beta RI. As a result, these type II receptors may share a common potential to signal via T beta RI. hGMR-RSK chimeric receptors may be useful tools for the identification and characterization of the divergent signals mediated by individual RSKs.     

Effects of N- and L-type calcium channel antagonists and (+/-)-Bay K8644 on nerve-induced catecholamine secretion from bovine perfused adrenal glands

Most proteins encoded by members of the Ly-49 gene family are class I-recognizing receptors on murine natural killer (NK) cells. Class I recognition by Ly-49 receptors usually results in inhibition of NK cell lysis of target cells. However, NK cells function not only in a lytic capacity, but also can mediate cytokine production. In this report we have demonstrated the ability of Ly-49A and Ly-49G2 to inhibit production of cytokines by NK cells by showing that specific antibodies against these gene products stimulate cytokine production. Murine NK cells were cultured in the presence of P815 (H2-Dd), and supernatants were analyzed for the production of interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocytemacrophage colony-stimulating factor (GM-CSF). NK cell populations were sorted for Ly-49A+ or Ly-49G+ subsets, and these subsets were analyzed for their ability to alter cytokine induction by target cell interaction. In the presence of target cells expressing the appropriate class I molecules, Ly-49A and G2 were found to inhibit cytokine induction by NK cells. Examination of mRNA for IFN-gamma and GM-CSF indicated that Ly-49 receptors increased mRNA levels of NK cells. These results demonstrate that class I binding of these NK receptors can inhibit production of important physiological cytokines, in addition to the regulation of cytotoxic activity.     

Modulation of life and death by the TNF receptor superfamily

The tumor necrosis factor receptor (TNFR) superfamily represents a growing family, with over 20 members having been identified thus far in mammalian cells. These proteins share significant homologies in their extracellular ligand binding domains and intracellular effector (death) domains. These receptors appear to transmit their signals via protein-protein interactions, which convey either a death or survival signal. Isolation and characterization of death domain containing proteins (TRADD, FADD/MORT-1, RIP), TRAF domain containing proteins (TRAF1-6) as well as new members and adaptor proteins such as DAXX have provided new insights to our understanding of signaling mechanisms associated with this family of receptors. While the death signals seem to be associated with the activation of both the caspase and JUN kinase pathways, the survival signals are mediated via the activation of the NF-kappaB pathway.     

Brain mapping in animals and humans

The discovery of the Mothers against dpp (Mad) gene in Drosophila has opened a window on an entirely unique signalling pathway that functions to mediate responses to the tumour growth factor beta (TGF beta) superfamily. This pathway, which is comprised of a family of proteins related to Mad, acts to convey signals directly from TGF beta receptors to the nucleus and is implicated in the pathogenesis of human diseases.