Radiation Hybrid Mapping of the Two Highly Homologous Human–Variant pMCHL Genes by PCR–SSCP

When gene loci are very similar in sequence, as in gene families or multiple pseudogenes, it is difficult to determine the specific location of the individual genes. We show here that applying PCR–SSCP to a radiation hybrid panel allowed mapping and specific sequencing of two genes with only a few sequence differences. Human–variant forms of the promelanin-concentrating hormone (pMCH) gene are found in two locations in the genome, previously localized by FISH to 5p14 and 5q12–q13. Without prior knowledge of sequence variation between the loci, we observed a difference in migration pattern in PCR–SSCP, indicating the presence of at least one point of sequence divergence. PCR–SSCP of 93 samples from a human–hamster radiation hybrid panel revealed the location of the genes to be between markers WI-4804 and AFM225YC5 on chromosome 5p, and between markers WI-3133 and WI-4225 on chromosome 5q. Sequencing of the two 680-bp PCR products from the hybrid panel demonstrated 3 bases of sequence difference between the 5p and 5q locations.     

Establishment and characterization of a mutagenized cell line exhibiting the ‘cell‐in‐cell’ phenotype at a high frequency

Cell‐in‐cell structures represent live cell events in which one cell internalizes another. Because formation of cell‐in‐cell structures is a rare event in most cell types and the event is associated with cell death, there has been limited clarification of this phenomenon, and its physiological role and molecular mechanism are yet to be precisely elucidated. In this study, we established a mutagenized cell line that exhibited cell‐in‐cell structures at a more than 10‐fold higher frequency as compared to the parent cells. Interestingly, both engulfment and invasion were increased in the mutagenized cell line as compared with that in the parent cell line in the suspension culture condition. This finding indicates that this mutagenized cell line showed an interchangeable status in terms of its ability to form cell‐in‐cell structures, and the system described here could be useful for elucidation of the mechanisms regulating the formation of cell‐in‐cell structures, including engulfment and invasion, in a given cellular environment. Further studies using this cell line are warranted to understand the mechanism of formation and biological significance of the cell‐in‐cell formation.     

Integrin–fibronectin interactions at the cell-material interface: initial integrin binding and signaling

Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor–ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of α₅β₁ integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor–ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin–Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling.     

NF1 loss disrupts Schwann cell–axonal interactions: a novel role for semaphorin 4F

Neurofibromatosis type 1 (NF1) patients develop neurofibromas, tumors of Schwann cell origin, as a result of loss of the Ras-GAP neurofibromin. In normal nerves, Schwann cells are found tightly associated with axons, while loss of axonal contact is a frequent and important early event in neurofibroma development. However, the molecular basis of this physical interaction or how it is disrupted in cancer remains unclear. Here we show that loss of neurofibromin in Schwann cells is sufficient to disrupt Schwann cell/axonal interactions via up-regulation of the Ras/Raf/ERK signaling pathway. Importantly, we identify down-regulation of semaphorin 4F (Sema4F) as the molecular mechanism responsible for the Ras-mediated loss of interactions. In heterotypic cocultures, Sema4F knockdown induced Schwann cell proliferation by relieving axonal contact-inhibitory signals, providing a mechanism through which loss of axonal contact contributes to tumorigenesis. Importantly, Sema4F levels were strongly reduced in a panel of human neurofibromas, confirming the relevance of these findings to the human disease. This work identifies a novel role for the guidance-molecules semaphorins in the mediation of Schwann cell/axonal interactions, and provides a molecular mechanism by which heterotypic cell–cell contacts control cell proliferation and suppress tumorigenesis. Finally, it provides a new approach for the development of therapies for NF1.     

Amino acid–amino acid contacts at the cooperativity interface of the bacteriophage λ and P22 repressors

The bacteriophage λ repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites. This cooperativity is mediated by a protein–protein interaction between the DNA-bound dimers. Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer–dimer interface. One of these pairs is nonconserved in the aligned sequences of the λ and P22 repressors; we show that a λ repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor. The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both λcI and P22c2 dimers.     

mTOR signaling at the crossroads of environmental signals and T-cell fate decisions

The evolutionarily conserved serine/threonine kinase mTOR (mechanistic target of rapamycin) forms the distinct protein complexes mTORC1 and mTORC2 and integrates signals from the environment to coordinate downstream signaling events and various cellular processes. T cells rely on mTOR activity for their development and to establish their homeostasis and functional fitness. Here, we review recent progress in our understanding of the upstream signaling and downstream targets of mTOR. We also provide an updated overview of the roles of mTOR in T-cell development, homeostasis, activation, and effector-cell fate decisions, as well as its important impacts on the suppressive activity of regulatory T cells. Moreover, we summarize the emerging roles of mTOR in T-cell exhaustion and transdifferentiation. A better understanding of the contribution of mTOR to T-cell fate decisions will ultimately aid in the therapeutic targeting of mTOR in human disease.     

Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling

Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor-ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of alpha5beta1 integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor-ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin-Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling.     

NK cell‐extrinsic IL‐18 signaling is required for efficient NK‐cell activation by vaccinia virus

NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR‐dependent and ‐independent pathways, as well as the NKG2D activating receptor that recognizes host stress‐induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study using C57BL/6 mice, we first showed that IL‐18 is critical for NK‐cell activation and viral clearance. We then demonstrated that IL‐18 signaling on both NK cells and DCs is required for efficient NK‐cell activation upon VV infection in vitro. We further showed in vivo that efficient NK‐cell activation in response to VV is dependent on DCs and IL‐18 signaling in non‐NK cells, suggesting an essential role for NK cell‐extrinsic IL‐18 signaling in NK‐cell activation. Mechanistically, IL‐18 signaling in DCs promotes expression of Rae‐1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell‐extrinsic IL‐18 signaling in NK‐cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK‐cell‐based therapies for viral infections and cancer.     

Methods to promote Notch signaling at the biomaterial interface and evaluation in a rafted organ culture model

The Notch signaling pathway is a promising target for controlling cell fate choices at the biomaterial-tissue interface. Building on our previous work in developing Notch-signaling biomaterials, we evaluated various immobilization schemes for Notch ligands, and their effect on human foreskin keratinocytes. A peptide sequence derived from the Jagged-1 DSL-region and immobilized to poly(2-hydroxyethyl methacrylate) (polyHEMA) showed no bioactivity in relation to the Notch-CSL pathway. The full-length Jagged-1 protein immobilized directly to the polyHEMA surface showed activity in signaling the Notch-CSL pathway. However, an indirect affinity immobilization approach yielded a stronger signal. Human keratinocytes plated on bound Jagged-1 showed upregulated involucrin, keratin 10, and loricrin protein expression, with this expression being cell density-dependent. Utilizing a human foreskin rafted organ culture model as a bridge between in vitro and in vivo studies, Jagged-1-modified or control polyHEMA rods were implanted in human foreskin and cultured at the air-medium interface. Keratinocyte proliferation was suppressed and intermediate-stage differentiation promoted in Jagged-1-modified rods compared with control rods. Thus, Notch-signaling biomaterials provide a robust approach to control keratinocyte differentiation and may find application to other progenitor and stem cells.     

Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface

Upon spinal cord injury, the central nervous system axons are unable to regenerate, partially due to the repulsive action of myelin inhibitors, such as the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). These inhibitors bind and signal through a single receptor/co-receptor complex that comprises of NgR1/LINGO-1 and either p75 or TROY, triggering intracellular downstream signaling that impedes the re-growth of axons. Structure–function analysis of myelin inhibitors and their neuronal receptors, particularly the NgRs, have provided novel information regarding the molecular details of the inhibitor/receptor/co-receptor interactions. Structural and biochemical studies have revealed the architecture of many of these proteins and identified the molecular regions important for assembly of the inhibitory signaling complexes. It was also recently shown that gangliosides, such as GT1b, mediate receptor/co-receptor binding. In this review, we highlight these studies and summarize our current understanding of the multi-protein cell-surface complexes mediating inhibitory signaling events at the neuron/myelin interface.     

T-cell influence on the B-cell differentiation process induced by Klebsiella

Klebsiella pneumoniae K43 cell membrane preparations (Klebs M) have been characterized previously as a human polyclonal B cell activator (PBA) that stimulates purified B cells to differentiate into immunoglobulin (Ig) secreting cells with negligible prior or parallel proliferation and in the absence of T cells. The aim of the present study was to define the cellular interactions in the regulation of Klebs M induced B-cell differentiation. For this purpose OKT4+ and OKT8+ cell populations were negatively selected with reasonable purity by means of a panning technique or by complement-mediated cytolysis using monoclonal OKT4 and OKT8 antibodies. The resulting cell populations were added to purified autologous B cells exposed to Klebs M or, as a control, pokeweed mitogen (PWM). In the Klebs M system both the OKT4+ and the OKT8+ cell subsets markedly enhanced IgM production; however, the helper effect of the OKT4+ cell subset was much more intense than that of the OKT8+ subset. In the PWM system only the OKT4+ cells provided help for B-cell differentiation. The OKT8+ subset demonstrated suppressor activity in the presence of an adequate helper cell (OKT4+ subset) function. These results indicate that Klebs M behaves like a "relatively T cell-independent PBA".     

Regulation of T-cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering signalosome assembly and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its ‘scaffolding’ function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation.     

Endothelial cell expression of galectin-1 induced by prostate cancer cells inhibits T-cell transendothelial migration

A critical control point in the immune response to tumors or to pathogens is the egress of lymphocytes from blood into damaged or infected tissue. While several specific endothelial cell proteins promote lymphocyte adhesion to and migration across endothelium, little is known about endothelial cell surface proteins that negatively regulate transendothelial migration of lymphocytes. Galectin-1 is a mammalian lectin expressed by a variety of cell types, including endothelial cells, that has pleiotropic anti-inflammatory effects. Galectin-1 is known to alter T-cell cytokine production and to trigger T-cell death. We now demonstrate that galectin-1 inhibits T-cell migration across endothelial cells, identifying a novel anti-inflammatory effect of galectin-1. We observed reduced T-cell migration across endothelial cells induced to increase galectin-1 expression by exposure to prostate cancer cell conditioned medium, compared to T-cell migration across control-treated endothelial cells, and the inhibitory effect of galectin-1 on T-cell migration was reversed by specific antiserum. Decreased T-cell migration was not due to decreased adhesion to galectin-1 expressing endothelial cells, nor to death of T cells, as T cells lacking core 2 O-glycans and thus resistant to galectin-1 death displayed reduced migration across endothelial cells. Galectin-1 on the surface of extracellular matrix also reduced the ability of T cells to migrate through the matrix. T cells bound to galectin-1-coated matrix demonstrated enhanced clustering of CD43, including at the T-cell:matrix interface, compared to CD43 on T cells bound to matrix in the absence of galectin-1. As translocation of CD43 to the trailing edge is essential for polarized T-cell migration, these data indicate that galectin-1-mediated clustering of CD43 contributes to the inhibitory effect on T-cell migration. Inhibition of T-cell migration is a novel anti-inflammatory activity of galectin-1.     

Sensitization of human breast cancer cells to natural killer cell‐mediated cytotoxicity by proteasome inhibition

The proteasome inhibitor, bortezomib, has direct anti‐tumour effects and has been demonstrated to sensitize tumour cells to tumour necrosis factor‐related apoptosis‐inducing ligand‐mediated apoptosis. Natural killer (NK) cells are effective mediators of anti‐tumour responses, both through cytotoxic granule killing and apoptosis‐inducing pathways. We therefore investigated if bortezomib sensitized human breast cancer cells to killing by the human NK cell line, NK‐92. Bortezomib was unable to sensitize MDA‐231 breast cancer cells to NK cell‐mediated killing in short‐term in vitro assays. However, bortezomib did cause these cells to up‐regulate apoptosis‐related mRNA as well as death receptors on the cell surface. In a long‐term in vitro tumour outgrowth assay that allows NK cells to use their full repertoire of killing pathways, bortezomib sensitized three breast cancer cell lines to NK cell‐mediated killing, which led to greater anti‐tumour effects than either treatment alone. We then used a xenogeneic mouse model in which CB‐17 SCID mice were injected with human breast cancer cells. This model displayed the effectiveness of NK‐92 cells, but the addition of bortezomib did not increase the survival further or reduce the number of lung metastases in tumour‐bearing mice. However, while bortezomib was highly cytotoxic to NK‐92 cells in vitro, bortezomib treatment in vivo did not decrease NK‐92 function, suggesting that through alternative dosing or timing of bortezomib, greater efficacy may occur from combined therapy. These data demonstrate that combined treatment of human breast cancer with bortezomib and NK cells has the potential to generate superior anti‐tumour responses than either therapy alone.