Design, synthesis and structure-activity relationship of simple bis-amides as potent inhibitors of GlyT1

Several novel classes of potent and small amide-type inhibitors of glycine transport (GlyT1) were developed through sequential simplification of a benzodiazepinone-lead structure identified from a high-throughput screening. The most potent compounds of these structurally simple classes show low nanomolar inhibition at the GlyT1 target.     

Mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41.

The mechanism of heparan sulfate (HS)-mediated human immunodeficiency virus type 1 (HIV-1) binding to and infection of T cells was investigated with a clone (H9h) of the T-cell line H9 selected on the basis of its high level of cell surface CD4 expression. Semiquantitative PCR analysis revealed that enzymatic removal of cell surface HS by heparitinase resulted in a reduction of the amount of HIV-1 DNA present in H9h cells 4 h after exposure to virus. Assays of the binding of recombinant envelope proteins to H9h cells demonstrated a structural requirement for an oligomeric form of gp120/gp41 for HS-dependent binding to the cell surface. The ability of the HIV-1 envelope to bind simultaneously to HS and CD4 was shown by immunoprecipitation of HS with either antienvelope or anti-CD4 antibodies from 35SO4(2-)-labeled H9h cells that had been incubated with soluble gp140. Soluble HS blocked the binding of monoclonal antibodies that recognize the V3 and C4 domains of the envelope protein to the surface of H9 cells chronically infected with HIV-1IIIB. The V3 domain was shown to be the major site of envelope-HS interaction by examining the effects of both antienvelope monoclonal antibodies and heparitinase on the binding of soluble gp140 to H9h cells.     

Amino-terminal processing of MIP-1beta/CCL4 by CD26/dipeptidyl-peptidase IV

CD26 is a membrane-bound ectopeptidase with dipeptidyl peptidase IV (DPPIV) activity that has diverse functional properties in T cell physiology and in regulation of bioactive peptides. We have previously reported that activated human peripheral lymphocytes (PBL) secrete an amino-terminal truncated form of macrophage inflammatory protein (MIP)-1beta/(3-69) with novel functional specificity for CCR1, 2, and 5. In this report, we show that the full length MIP-1beta is processed by CD26/DPPIV to the truncated form and that cleavage can be blocked by DPPIV inhibitory peptides derived from HIV Tat(1-9) or the thromboxane A2 receptor, TAX2-R(1-9). Addition of Tat(1-9) or TAX2-R(1-9) peptides to PBL cultures partially blocks endogenous MIP-1beta processing. The kinetics of conversion of MIP-1beta from intact to MIP-1beta(3-69) in activated PBLs correlates with cell surface expression of CD26. Our results suggest that NH2-terminal processing of MIP-1beta and possibly other chemokines may depend on the balance between CD26/DPPIV enzymatic activity and cellular and viral proteins that modulate enzyme function.     

Natural truncation of the chemokine MIP-1 beta /CCL4 affects receptor specificity but not anti-HIV-1 activity

Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

Identifying spawning behavior in Pacific halibut, <Emphasis Type="BoldItalic">Hippoglossus stenolepis</Emphasis>, using electronic tags

Synopsis Identifying spawning behavior in Pacific halibut, Hippoglossus stenolepis, is particularly challenging because they occupy a deep, remote environment during the spawning season. To identify spawning events, a method is needed in which direct observation by humans is not employed. Spawning behavior of seven other flatfish, species has been directly observed in their natural environment by investigators using SCUBA. All of these flatfish species display almost identical spawning behavior that follows a routine. Therefore, it is reasonable to believe that this spawning behavior occurs in other flatfish species, including Pacific halibut. As part of a larger study, we recaptured two Pacific halibut on which Pop-up Archival Transmitting (PAT) tags had been attached during the winter spawning season. Because the tags were physically retrieved, we were able to collect minute-by-minute depth records for 135 and 155 days. We used these depth data to tentatively identify spawning events. On seven separate occasions between 20 January 2001 and 9 February 2001, one fish displayed a conspicuous routine only seen during the spawning season of Pacific halibut and the routine parallels the actions of other spawning flatfish directly observed by humans using SCUBA. Therefore, we propose this routine represents spawning behavior in Pacific halibut. The second tagged fish did not display the conspicuous routine, thus challenging the assumption that Pacific halibut are annual spawners. PAT tags may prove to be a useful tool for identifying spawning events of Pacific halibut, and that knowledge may be used for improved management in the future.     

CpG-independent synergistic induction of beta-chemokines and a dendritic cell phenotype by orthophosphorothioate oligodeoxynucleotides and granulocyte-macrophage colony-stimulating factor in elutriated human primary monocytes

Chemokines attract leukocytes bearing the relevant chemokine receptors and regulate innate immune responses. CpG oligodeoxynucleotides (ODN) and GM-CSF are potent vaccine adjuvants and in combination induce enhanced Th1 responses by mechanisms yet to be determined. We have examined combinations of CpG- or non-CpG-ODN and GM-CSF for effects on the production of chemokines and the differentiation of monocytes to dendritic cells. High levels of the Th1-attracting, HIV-1-inhibitory chemokines, CCL3/MIP-1alpha and CCL4/MIP-1beta, were induced in human primary monocytes when CpG- or non-CpG-ODN was combined with GM-CSF, but not with IL-4 or IFN-gamma. The synergistic induction of beta-chemokines by non-CpG-ODN was phosphorothioate (PS) chemistry dependent and inhibited by blocking endosome maturation/acidification and ERK1/2 activation. Chemokine and TLR9 mRNAs were induced by PS-ODN. Cells treated with non-CpG PS-ODN and GM-CSF expressed dendritic cell marker CD83 and high levels of HLA-DR and costimulatory molecules, and were CD14(-) or CD14(dim), consistent with monocyte differentiation into a dendritic cell phenotype. The induction of CD83 and beta-chemokines was tyrosine phosphorylation dependent. Secreted CCL3 and CCL4 were detected as a heterodimer. Our results indicate the CpG-independent synergy between PS-ODN and GM-CSF mediated through chemokine and dendritic cell induction. In addition, our observations suggest that PS-ODN plus GM-CSF may be useful as potent ex vivo dendritic cell differentiation/maturation agents for dendritic cell therapy and as vaccine adjuvants for tumor and infectious microorganisms, including HIV-1.     

A somatic cell mutant with a kinetically altered 3-hydroxy-3-methylglutaryl coenzyme A reductase

Deinococcus radiophilus strain ATCC 27603 contains, apart from endonuclease DraI [1], two additional sequence-specific endonucleases. These enzymes, designated DraII and DraIII, recognize nucleotide sequences with novel specificities, PuG↓GNCCPy and CACNN↓GTG, respectively.