Lymphocyte surface receptors and albumin

Albumin was shown to be hidden component of mouse B and T lymphocyte plasma membranes. It was readily radiolabeled from within the plasma membrane by a lipophilic, photoactivated reagent (125I-iodonaphthylazide) but not from the cell exterior by lactoperoxidase-catalyzed iodination; it was not detected by immunofluorescence on intact cells. The function of this cryptic lymphocyte membrane albumin is unknown at present. This cryptic albumin component was discovered during immunoprecipitation experiments using anti-Ig reagents. It is considered of general methodologic significance that many antisera, both native and rigorously Ig-absorbed (both positively and negatively), contained such contaminating activity. The possibility is raised that such contaminating activity may be involved in some reported findings of albumin-size "Ig-like heavy chains" in both the B and T lymphocyte lineages.     

Hybridization triggered cross-linking of deoxyoligonucleotides.

This paper reports details of the synthesis of oligodeoxynucleotides containing the modified base 5-methyl-N4,N4-ethanocytosine (Ce). The 9-fluorenylmethoxycarbonyl group is used as a protecting group for the exocyclic amines of dA and dC. This group can be removed rapidly under very mild conditions. Oligomers containing the Ce base form a cross-link when hybridized to their complementary deoxyoligonucleotides. Some of the scope and limitations of these cross-link forming oligonucleotides are reported.     

Lymphocyte homing receptors and the immune response in vivo

An important aspect of the developmental maturation of lymphocytes is their capacity to locate and enter lymphoid organs with great rapidity and specificity and to follow certain routes within these organs for the attainment of particular immunological capabilities. It is now known that this “homing response” to lymphoid organs involves specific glycoprotein receptors on the lymphocyte cell surface. The biochemistry of these receptors and their significance in normal and pathological immune responses are discussed.     

Induction of IL-5 receptors on normal B cells by cross-linking surface Ig with anti-Ig-dextran.

Regulation of IL-5R expression in normal, non-Ly-1 (CD5) B cells was evaluated. Freshly isolated unfractionated spleen B cells express little or no detectable IL-5R. In contrast, B cells stimulated with anti-Ig-dextran conjugates express substantial numbers of IL-5R. Phenotypic analysis of the B cells responding to anti-Ig-dextran, and expressing IL-5R, demonstrates that these cells do not express Ly-1 or Mac-1. Scatchard analysis of B cells stimulated with anti-IgD-dextran reveals two classes of IL-5R: a high affinity receptor with a Kd of 17 pM and approximately 300 receptors/cell, and a low affinity receptor with a Kd of 0.6 nM and approximately 1000 receptors/cell. Peak receptor expression in response to anti-IgD-dextran is seen 72 h after stimulation and with a dose of 10 ng/ml. The induced receptors are functional, because both proliferation and Ig secretion by B cells treated with anti-IgD-dextran are enhanced by IL-5. Other B cell mitogens such as LPS, soluble anti-Ig/IL-4, or phorbol esters/ionomycin are poor inducers of the IL-5R. Finally, not only does LPS fail to induce significant IL-5R expression on spleen B cells, it suppresses both high and low affinity IL-5R expression induced by anti-IgD-dextran. These data indicate that normal, non-Ly-1 B cells are capable of expressing both high and low affinity IL-5R but that receptor expression is critically dependent on the type of stimulus provided to the B cell. A stimulus that produces extensive cross-linking of surface Ig on B cells, i.e., anti-Ig-dextran, is very effective in inducing IL-5R whereas a variety of other B cell mitogens are ineffective.     

Interferon (IFN)-like antiviral effect is induced by unspecific cross-linking of cell surface receptors

Photosystem I preparations were irradiated with UV to destroy vitamin K₁ in situ. The depletion of vitamin K₁ resulted in inactivation of NADP⁺ photoreduction and introduction of a 220 ms component in the flash generated P700⁺ re-reduction at room temperature. The photoreduction of the terminal FeS centers FA and FB in control and vitamin K₁-depleted preparations at 7 K were comparable. The data confirm that vitamin K₁ is functionally implicated in primary electron transfer reactions in PS I at physiological temperature, and that the anomalous results at cryogenic temperature may be explicable in terms of a by-pass of the vitamin K₁ acceptor site or heterogeneity introduced into the photosystem by quinone removal.     

Lymphocyte alpha-actinin. Relationship to cell membrane and co-capping with surface receptors

Mouse spleen lymphocytes synthesize a protein which comigrates with skeletal muscle alpha-actinin on two-dimensional gel electrophoresis and is immunoprecipitated by an antibody directed against skeletal muscle alpha-actinin. Mouse lymphocyte alpha-actinin is present in membrane fractions, and is immunoprecipitated from lymphocyte detergent lysates by an antiserum made against these purified membranes. The anti- alpha-actinin activity of this antiserum is not adsorbed after incubation with fixed intact lymphocytes. Lymphocyte alpha-actinin does not bind concanavalin A and it is inaccessible to lactoperoxidase- catalyzed surface iodination. Double immunofluorescence shows that alpha-actinin moves concurrently along the cell membrane with redistributed surface immunoglobulins and Thy-1 antigen, and remains associated up to 30 min with surface aggregates of these receptors. Our results suggest that lymphocyte alpha-actinin, as defined by molecular weight and cross reactivity with the antibody against the muscle protein, (a) is associated with the cell membrane, (b) is not expressed at the cell surface, and (c) participates in the movement of surface receptors.     

DNA-damage response pathways triggered by viral replication

Many viruses, with distinct replication strategies, activate DNA-damage response pathways, including the lentivirus human immunodeficiency virus (HIV) and the DNA viruses Epstein-Barr virus (EBV), herpes simplex virus 1, adenovirus and SV40. DNA-damage response pathways involving DNA-dependent protein kinase, ataxia-telengiectasia mutated (ATM) and 'ataxia-telengiectasia and Rad3-related' (ATR) have all been implicated. This review focuses on the effects of HIV and EBV replication on DNA repair pathways. It has been suggested that activation of cellular DNA repair and recombination enzymes is beneficial for viral replication, as illustrated by the ability of suppressors of the ATM and ATR family to inhibit HIV replication. However, activation of DNA-damage response pathways can also promote apoptosis. Viruses can tailor the cellular response by suppressing downstream signalling from DNA-damage sensors, as exemplified by EBV. New small-molecule inhibitors of the DNA-damage response pathways could therefore be of value to treat viral infections.     

Chemical cross-linking of heteromeric glucocorticoid receptors

Glucocorticoid receptors of wild-type and nti ("increased nuclear transfer") mutant S49.1 mouse lymphoma cells exist in extracts under low-salt conditions predominantly as high molecular weight species (Mr greater than or equal to 300,000). These receptor-hormone complexes are unable to bind to DNA. High salt (300 mM KCl) produces dissociated receptors of Mr 116,000 and 60-A Stokes radius (wild type) and Mr 60,000 and 38-A Stokes radius (nti mutant), both of which bind to DNA. We used reaction with bifunctional N-hydroxysuccinimide esters as well as oxidation with Cu2+/o-phenanthroline to stabilize the high molecular weight structures. These cross-linked complexes do not interact with DNA, but reductive cleavage again produces the dissociable receptor forms and restores their ability to bind to DNA. The protein modifying reagents iodoacetamide and diethyl pyrocarbonate also produce stabilized high molecular weight receptor complexes. Cross-linking of the high molecular weight receptor forms can also be achieved in intact cells. Immunochemical techniques were used to prove that the complexes cross-linked either in vivo or in cell extracts do contain the heat shock protein of Mr 90,000 as a common constituent. The data show that the high molecular weight receptor complexes are preexisting in intact cells and that dissociation generates DNA binding ability.     

Design and synthesis of the novel cross-linking reagents triggered by the triple helix formation.

In our attempt to new nucleobase analogs capable of interstrand cross-linking, we developed 2-amino-6-vinyl purine analog (1). The oligonucleotides incorporating 1 showed efficient interstrand cross-linking with selectivity toward cytidine at a target site. In this paper, we describe the design of the new cross-linking reagents (2) bearing 2-amino-6-vinyl purine motif, and triplex-directed alkylation with 2 to double-stranded DNA.     

Neutrophil chemotaxis and superoxide production are induced by cross-linking FcgammaRII receptors

Neutrophils express two types of receptor for the Fc region of IgG, FcgammaRII and FcgammaRIIIB. Via these receptors, neutrophils bind IgG complexes that contain more than one IgG molecule. This binding activates functional processes, such as the respiratory burst and chemotaxis. Neutrophils were treated with biotinylated anti-Fc receptor monoclonal antibodies and chemotaxis toward streptavidin, a cross-linking agent, was determined. Cross-linking FcgammaRII and not FcgammaRIIIB induced neutrophil chemotaxis. Superoxide production in response to immobilized anti-Fc receptor antibodies was also examined. Anti-FcgammaRII Fab bound to ELISA plates induced superoxide production, while anti-FcgammaRIIIB Fab did not. Pretreatment of neutrophils with anti-FcgammaRII Fab reduced superoxide generated by immobilized anti-FcgammaRII antibody. The data demonstrate that FcgammaRII and not FcgammaRIIIB are responsible for neutrophil chemotaxis and superoxide production upon Fc receptor activation.     

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.     

Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking

The structure of receptors for insulin-like growth factors in rat liver plasma membranes and the BRL 3A2 rat liver cell line has been examined by chemical cross-linking with disuccinimidyl suberate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Two receptor subtypes have been identified: (i) 125I multiplication-stimulating activity cross-linked to liver membranes or intact cells appeared in a complex of Mr = 260,000 (reduced) and 220,000 (nonreduced) and (ii) 125I-insulin-like growth factor I cross-linked to BRL 3A2 cells appeared predominantly in two bands of Mr greater than 300,000 without disulfide reduction and in a Mr = 130,000 complex following reduction. The two subtypes of insulin-like growth factor receptors identified by structural analysis correspond to previously observed differences in their specificity for insulin and insulin-like growth factors.     

A novel cell response triggered by interphase centromere structural instability

Interphase centromeres are crucial domains for the proper assembly of kinetochores at the onset of mitosis. However, it is not known whether the centromere structure is under tight control during interphase. This study uses the peculiar property of the infected cell protein 0 of herpes simplex virus type 1 to induce centromeric structural damage, revealing a novel cell response triggered by centromere destabilization. It involves centromeric accumulation of the Cajal body-associated coilin and fibrillarin as well as the survival motor neuron proteins. The response, which we have termed interphase centromere damage response (iCDR), was observed in all tested human and mouse cells, indicative of a conserved mechanism. Knockdown cells for several constitutive centromere proteins have shown that the loss of centromeric protein B provokes the centromeric accumulation of coilin. We propose that the iCDR is part of a novel safeguard mechanism that is dedicated to maintaining interphase centromeres compatible with the correct assembly of kinetochores, microtubule binding, and completion of mitosis.