SFR3-DR5, a monoclonal antibody with HLA-DR5 specificity

We have developed a monoclonal antibody with HLA-DR5 serologic specificity. The antibody, SFR3-DR5, binds specifically to DR5-positive lymphoblastoid cell lines, and immunoprecipitates alpha- and beta-chains characteristic of DR antigens from them. Cytotoxic activity of the antibody segregates with the DR5-bearing haplotype in a family. The antibody reacted with the cells of 16 of 17 DR5 individuals and was negative on all DR5-negative cells tested. SFR3-DR5 reacted weakly with PWM-activated cells of the single DR5 individual whose B lymphocytes were unreactive with the monoclonal antibody by cytotoxicity. Possible interpretations of these results are discussed.     

MDA-5 recognition of a murine norovirus

Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5-/- dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5-/- DCs as well as in MDA5-/- mice in vivo. Interestingly, TLR3-/- DCs do not have a defect in vitro, but TLR3-/- mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.     

Modeling molecular mechanisms of binding of the anaphylatoxin C5a to the C5a receptor

This study presents the 3D model of the complex between the anaphylatoxin C5a and its specific receptor, C5aR. This is the first 3D model of a G-protein-coupled receptor (GPCR) complex with a peptide ligand deduced by a molecular modeling procedure analyzing various conformational possibilities of the extracellular loops and the N-terminal segment of the GPCR. The modeling results indicated two very different ways of interacting between C5a and C5aR at the two interaction sites suggested earlier based on the data of site-directed mutagenesis. Specifically, C5a and C5aR can be involved in "mutual-induced fit", where the interface between the molecules is determined by both the receptor and the ligand. The rigid core of the C5a ligand selects the proper conformations of the highly flexible N-terminal segment of C5aR (the first interaction site). At the same time, the binding conformation of the flexible C-terminal fragment of C5a is selected by well-defined interactions with the TM region of the C5aR receptor (the second interaction site). The proposed 3D model of C5a/C5aR complex was built without direct use of structural constraints derived from site-directed mutagenesis reserving those data for validation of the model. The available data of site-directed mutagenesis of C5a and C5aR were successfully rationalized with the help of the model. Also, the modeling results predicted that the full-length C5a and C5a-des74 metabolite would have different binding modes with C5aR. Modeling approaches employed in this study are readily applicable for studies of molecular mechanisms of binding of other polypeptide ligands to their specific GPCRs.     

Human T cells express the C5a receptor and are chemoattracted to C5a

The anaphylatoxin C5a is a potent mediator of inflammation that exerts a broad range of activity on cells of the myeloid lineage. In this study, we present the first evidence that human T cells express the C5a receptor (C5aR) and are chemotactic to C5a. Using FACS analysis, we found that the C5aR was expressed at a low basal level on unstimulated T cells and was strikingly up-regulated upon PHA stimulation in a time- and dose-dependent manner. CD3+ sorted T cells as well as Jurkat T cells were shown to express C5aR mRNA as assessed by RT-PCR. Moreover, semiquantitative RT-PCR analysis demonstrated that C5aR mRNA was down-regulated in purified T cells upon long-term PHA stimulation. To demonstrate that C5a was biologically active on T cells, we investigated the chemotactic activity of C5a and observed that purified CD3+ T cells are chemotactic to C5a at nanomolar concentrations. Finally, using a combination of in situ hybridization and immunohistochemistry, we showed that the T cells infiltrating the central nervous system during experimental allergic encephalomyelitis express the C5aR mRNA. In summary, these results suggest that C5a exerts direct effects on T cells and could be involved in the trafficking of T cells under physiological and pathological conditions, including inflammatory diseases of the central nervous system.     

Binding thermodynamics of serotonin to rat-brain 5-HT1a, 5HT2a and 5-HT3 receptors

The thermodynamic parameters ΔG° , ΔH° and Δs° of the binding equilibrium of serotonin to 5-HT_1A, 5-HT_2A and 5-HT₃ rat-brain membrane receptors have been determined by means of affinity constant measurements at six temperatures in the range 0 –35 ° C and van't Hoff plots. At variance with 5-HT_1A and 5-HT₃, the binding at the 5-HT_2A receptors is strongly endothermic and entropy-driven. Comparison with the results obtained by other authors on 5-HT_2A receptors in rats and humans suggests that the observed differences can be explained by a single amino acid difference in the receptor sequence between these two species.     

Potent inhibition of the hypothalamic progesterone 5 alpha-reductase by a 5 alpha-dihydroprogesterone analog

The ability of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit the progesterone 5 alpha-reductase and the two 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADH- and NADPH-linked) from female rat hypothalamus has been studied. Dose response experiments indicate that AMPD is a potent antagonist of hypothalamic progesterone 5 alpha-reduction but is an ineffective inhibitor of the NADPH- and NADH-linked 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. Kinetic analyses of the interaction of AMPD with the progesterone 5 alpha-reductase show that it is a competitive inhibitor versus progesterone (Ki(slope) = 6.2 +/- 0.5 nM; apparent Km (progesterone) = 130 +/- 12 nM) and an uncompetitive inhibitor versus NADPH (Ki(intercept) = 11.8 +/- 0.8 nM). These inhibition patterns are consistent with the concept that NADPH binding precedes that of either AMPD or progesterone. The inhibition of the progesterone 5 alpha-reductase by AMPD does not appear irreversible since preincubation of the enzymatic activity (at 37 degrees C) with inhibitor and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. Furthermore, this inhibition can be easily removed via dilution, even following a 60-min preincubation with AMPD and NADPH. It is postulated that the specific and powerful inhibition of the progesterone 5 alpha-reductase by AMPD may be due to this compound functioning as a transition state analog. This inhibitor should prove valuable in studying the characteristics of the progesterone 5 alpha-reductase and the function of hypothalamic progestin metabolism.     

Study on the enzymatic 5′-deiodination of 3′,5′-diiodothyronine using a radioimmunoassay for 3′-iodothyronine

A radioimmunoassay for 3′-iodothyronine has been developed. All iodothyronine analogues (except 3,3′-diiodothyronine) showed very little (0.02% at most) cross-reactivity, and the assay was sensitive to 1 pg 3′-iodothyronine/ tube. We have studied the 5′-deiodination of 3′,5′-diiodothyronine by rat liver microsomal fraction in the presence of dithiothreitol. Production of 3′-iodothyronine at 37°C was found to be linear with time of incubation up to 30 min and with concentration of microsomal protein up to 100 μg/ml. The reaction rate reached a limit on increasing 3′,5′-diiodothyronine concentration to 10 μM. The effect of pH on 3′-iodothyronine production was found to depend on 3′,5′-diiodothyronine concentration. Increasing 3′,5′-diiodothyronine concentration from 0.1 to 10 μM resulted in a shift of the pH optimum from 6–6.5 to 7.5. Similar effects on the 5′-deiodination of 3,3′,5′-triiodothyronine were observed, supporting the hypothesis that these reactions are catalysed by a single enzyme (iodothyronine 5′-deiodinase).     

Apoptotic Cleavage of Rabaptin-5-like Proteins and a Model for Rabaptin-5 Inactivation in Apoptosis

Intracellular membrane transport from the plasma membrane is one of the processes affected in apoptotic cells. Apoptotic inhibition of endosomal transport occurs due to cleavage of Rabaptin-5, an effector of small GTPase Rab5, which results in inhibition of early endosome fusion. Recently several novel Rabaptin-5-like proteins were identified. We investigated whether Rabaptin-5-like proteins, Rabaptin-5? and Rabaptin-5?, are also cleaved in apoptosis and found that both proteins are cleaved in apoptotic cell extracts by caspase-3-related proteases. This suggests that functional inactivation of these proteins is necessary for apoptotic cell death. We also mapped a novel, N-terminal, putative Rab5 binding site in Rabaptin-5-like proteins, which becomes physically separated from the previously known C-terminal Rab5 binding site after apoptotic cleavage of these proteins. Presence of the second Rab5 binding site provides a new insight into Rabaptin-5 function in early endosome fusion and a mechanistic model for functional inactivation of Rabaptin-5 in apoptosis.     

Plexin-B3 is a functional receptor for semaphorin 5A

Semaphorins are a large family of molecular cues implicated in neural development and in a variety of functions outside the nervous system. Semaphorin 5A (Sema5A) is a transmembrane semaphorin, containing seven thrombospondin type-1 repeats, which was recently found to control axon guidance. Here we show that plexin-B3 is a high-affinity receptor specific for Sema5A. We further demonstrate that plexin-B3 activation by Sema5A mediates functional responses in plexin-B3-expressing cells (either fibroblasts, epithelial and primary endothelial cells). In addition, Sema5A can trigger the intracellular signalling of the hepatocyte growth factor/scatter factor receptor, Met, associated in a complex with plexin-B3. We thus conclude that Sema5A is able to elicit multiple functional responses through its receptor plexin-B3.     

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.     

C5a and C5a(desArg) enhance the susceptibility of monocyte-derived macrophages to HIV infection

Mononuclear phagocytes, which include circulating blood monocytes and differentiated tissue macrophages, are believed to play a central role in the sexual transmission of HIV infection. The ability of HIV to productively infect these cells may be influenced by action of exogenous or host-derived substances at the site of viral entry. Given the potent capacities of inflammatory mediators to stimulate anaphylatoxic and immunomodulatory functions in mucosa, the effects of complement-derived anaphylatoxins on the susceptibility of monocytes and monocyte-derived macrophages (MDM) to HIV-1 infection were examined. In our in vitro system, the susceptibility to infection was up to 40 times increased in MDM that had been exposed to C5a or C5a(desArg), but not to C3a or C3a(desArg), for 2 days before adding of virus. By contrast, the treatment with complement anaphylatoxins did not affect HIV replication in fresh monocytes. Stimulatory effect of C5a and its desArg derivative on HIV infection correlated with the increase of TNF-alpha and IL-6 secretion from MDM. All these functional effects of C5a and C5a(desArg) were reversible by treatment of cells with the mAb that functionally blocks C5aR. Taken together, these results indicate that C5a and C5a(desArg) may increase the susceptibility of MDM to HIV infection through stimulation of TNF-alpha and IL-6 secretion from these cells.     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

Synthesis and properties of 5'-triphosphoryl 2'-5' oligoadenylates (2-5A), and a general method for synthesis of 3', 5'-bisphosphorylated oligonucleotides.

2'-5'-Linked oligoadenylic acid 5'-triphosphates (2-5A) having chain lengths of 2-4 have been synthesized by polymerization of 3'-O-(o-nitrobenzyl)-N-benzoyladenosine 5'-phosphate followed by 5'-triphosphorylation via the imidazolidates. A large scale preparation of 5'-O-phosphoryladenylyl-(2'-5')-adenylyl-(2'-5')-adenosine was performed by the phosphotriester method using 5'-O-monomethoxytrityl-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate and 5'-O-phosphorodianilido-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate as intermediates. The trimer was also triphosphorylated by the imidazolide method. CD spectra for 5'-mono and triphosphorylated 2'-5' adenylates were measured as well as their UV hypochromicities. This triester method was also applied to the synthesis of 3',5'-bisphosphorylated protected oligoadenylic acids with natural 3'-5' linkages which could be used for further condensations to yield 5'-phosphorylated polynucleotides.     

Evaluating a Parainfluenza Virus 5-Based Vaccine in a Host with Pre-Existing Immunity against Parainfluenza Virus 5

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.     

Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CD5-regulated genes in a B cell line suggest a role for CD5 in malignant phenotype

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.     

Functional analysis of 2-5A-dependent RNase and 2-5a using 2',5'-oligoadenylate-cellulose

2-5A is an intracellular effector that has been implicated in interferon action, hormonal regulation, and cell growth control. 2-5A action is mediated through its activation of 2-5A-dependent RNase (RNase L, RNase F). Affinity resins [2-5A-cellulose and core (2-5A)-cellulose] were chemically synthesized for purification and immobilization of 2-5A-dependent RNase from mouse L cells and rabbit reticulocyte lysates. The breakdown of poly(U)-[3'-32P]Cp to acid-soluble fragments was demonstrated using the 2-5A-dependent RNase:2-5A -cellulose complex; this activity was enhanced by adding (free) 2-5A. In contrast, RNase activity was measured from the 2-5A-dependent RNase:core (2-5A)-cellulose complex only after the addition of free 2-5A. The rabbit reticulocyte 2-5A-dependent RNase is activated only by tetramer or higher oligomers of 2-5A; therefore there was breakdown of poly(U)-[3'-32P]Cp using core (2-5A)-cellulose-bound reticulocyte 2-5A-dependent RNase after addition of tetramer 2-5A but there was no poly(U) degradation in the presence of trimer 2-5A. The absence of significant general nuclease in the assays was demonstrated by the resistance to breakdown of poly(C)-[3'-32P]Cp (not susceptible to 2-5A-dependent RNase). Moreover, core (2-5A)-cellulose was used to develop a sensitive (subnanomolar) assay for the detection of authentic 2-5A. 2-5A, or the material to be tested, was added to mouse L-cell 2-5A-dependent RNase:core (2-5A)-cellulose complex in the presence of poly(U)-[3'-32P]Cp. The concentration of 2-5A in the sample could be measured from the amount of poly(U) degradation. Several closely related analogs of 2-5A were tested and found to be completely inactive. The technology described herein may be applied to the study of the regulation of 2-5A-dependent RNase, the detection of 2-5A from cells and tissues, and other aspects of the 2-5A system.