The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogenactivated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125 I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr 175 , which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a K m of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a K i of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125 I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

CD4 glycoprotein on the surface of T cells helps in the immune response and is the receptor for HIV infection. The structure of a soluble fragment of CD4 determined at 2.3 Å resolution reveals that the molecule has two intimately associated immunoglobulin-like domains. Residues implicated in HIV recognition by analysis of mutants and antibody binding are salient features in domain D1. Domain D2 is distinguished by a variation on the β-strand topologies of antibody domains and by an intra-sheet disulphide bridge.CD4, a cell-surface glycoprotein found primarily on T lymphocytes, is required to shape the T-cell repertoire during thymic development and to permit appropriate activation of mature T cells 1 . T cells that recognize antigens associated with class II major histocompatibility complex (MHC) molecules, mainly T helper cells, express CD4. Evidence is accumulating that CD4 and the T-cell receptor coordinately engage class II molecules on antigenpresenting cells to mediate an efficient cellular immune response, and that engaged CD4 may transmit a signal to an associated cytoplasmic tyrosine kinase, p56 lck .CD4 belongs to the immunoglobulin superfamily of molecules which generally serve in recognition processes 2,3 . The sequence of CD4 4,5 indicates that it consists of a large (~370 residues) extracellular segment composed of four tandem immunoglobulin-like domains, a single transmembrane span, and a short (38 residues) C-terminal cytoplasmic tail. The first domain (D1) shares several features with immunoglobulin variable domains, but the sequence similarities between immunoglobulins and the other extracellular domains (D2, D3 and D4) are more remote.In humans, CD4 can be subverted from its normal immuno-supportive role to become the receptor for infection by the human immunodeficiency virus (HIV) 1,6,7 . Recombinant soluble CD4 proteins bind to the HIV envelope glycoprotein gp120, and can thus inhibit viral infection and virus-mediated cell fusion in vitro (refs 8, 9 and references therein). (refs 21-23 and unpublished results), the main flexibility seems to be at the D2 to D3 junction. We have now crystallized a truncated derivative of CD4 that diffracts well, and here we report its atomic structure. This recombinant fragment 8 as secreted from Chinese hamster ovary (CHO) cells consists of residues 1-183 of human CD4 plus two missense residues, Asp-Thr; and it is unglycosylated. This molecule, which we refer to as D1D2, is as active as sCD4 in binding to gp120 (dissociation constant K d ≃ 3 nM) and retains all antibody epitopes mapped to these domains of CD4 (ref. 8 and unpublished results). Others have crystallized similar fragments from the N-terminal half of sCD4 24,25 and the structure of one is reported in the accompanying paper 25 . HHS Public AccessHere we describe the D1D2 structure in comparison with that of immunoglobulin domains, provide a geometrical definition for HIV recognition sites, and discuss implications of the structure for normal CD4 function and evolution of the immunoglobu...

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Seventeen malaria-naive volunteers received a recombinant Plasmodium falciparum vaccine (RLF) containing the carboxy- and the amino-terminal of the circumsporozoite protein (CSP) antigen without the central tetrapeptide repeats. The vaccine was formulated in liposomes with either a low or high dose of 3-deacylated monophosphoryl lipid A (MPL) and administered with alum by intramuscular injection. Both formulations were well tolerated and immunogenic. MPL increased sporozoite antibody titers measured by ELISA, Western blot, and immunofluorescence assay. One high-dose MPL vaccine formulation recipient developed a CSP-specific cytotoxic T lymphocyte response. After homologous sporozoite challenge, immunized volunteers developed patent malaria. There was no correlation between prepatent period and antibody titers to the amino- or carboxy-terminal. The absence of delay in patency argues against inclusion of the amino-terminal in future vaccines. A significant cytotoxic T lymphocyte response may have been suppressed by the inclusion of alum as an adjuvant.

Pig brain contains three forms of glutamate decarboxylase with pI values of 5.3, 5.5 and 5.8, referred to as the alpha-, beta- and gamma-forms respectively. These forms were purified and kinetically characterized. The major synaptic form of glutamate decarboxylase (the beta-form) migrated as a single band on electrophoresis in sodium dodecyl sulphate/polyacrylamide gels with an apparent Mr of 60 000. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by immunoblotting with an affinity-purified antibody to the enzyme indicated a subunit Mr of 60 000 for the alpha- and gamma-forms as well. An extensive kinetic analysis, aided by an integrated equation that describes the inactivation and re-activation cycle of the enzyme, revealed that the three forms of the enzyme differ markedly in kinetic properties. The Km values for L-glutamate were 0.17, 0.45 and 1.24 mM respectively for the alpha-, beta- and gamma-forms. The Ki for 4-aminobutyrate, the first-order rate constants for inactivation by L-glutamate and 4-aminobutyrate, the rate constant for re-activation of the apoenzyme by pyridoxal 5'-phosphate and the dissociation constant for pyridoxal 5'-phosphate also differed in a similar way among the three forms; the values were in the order alpha-form less than beta-form less than gamma-form.