SummaryThe first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A 2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, LYS46, and Arg59, on the C, C', C", and D strands, respectivdy. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gpi20. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction.T he CD4 transmembrane glycoprotein participates in key steps of thymocyte differentiation (1-3) and T lymphocyte activation (4-9) by interacting with nonpolymorphic regions of class II MHC molecules (10--12). It is expressed on most thymocytes (1) and a subset of T lymphocytes, including helper T cells and class II MHC-specific cytotoxic T cells (5-7, D, 14). CD4 in humans is also the receptor for HIV (15-18). It is required for viral attachment and subsequent entry into ceils (19), and these functions account for HIV T cell tropism. The virus binds through a contact made by the gp120 component of its envelope glycoprotein (20). The binding of gp120 to CD4 has been implicated in the cellular depletion of CD4 + lymphocytes in AIDS (21-24), and circulating free gp120 is thought to interfere with normal immune function (12, 25). The central role of the CD4-gp120 interaction in the pathogenesis of AIDS justifies a detailed analysis in molecular terms, and inhibition of that interaction is also an attractive target for antiviral intervention.The extracellular part of CD4 contains four Ig-like do= mains (26). The gp120 binding site lies in the most NH2 terminal of these domains (27,28). A number of studies eraploying escape mutants (29), insertion mutagenesis (30), homology scanning mutagenesis (12,28,31), and alanine scanning mutagenesis (32) have helped to restrict the binding site to a segment surrounding the region homologous to the CDR2 of an Ig V domain.The atomic structure of a fragment containing the first two domains of CD4 has been determined by x-ray crystallography (33, 34). The first domain contains two antiparallel sheets, as predicted by its Ig V homology. The mutations that affect gp120 binding lie mostly in the antiparalld strands, denoted C' and C", that form a ridge at one edge of the domain as well as in the adjacent D strand. The C'C" loop is longer than the CDR2 loop of a typical Ig V domain, and it projects prominently ...
From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.
The alkyl chain length of quaternary ammonium/PEG copolyoxetanes has been varied to discern effects on solution antimicrobial efficacy, hemolytic activity and cytotoxicity. Monomers 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) were used to prepare precursor P[(BBOx)(ME2Ox)-50:50–4 kDa] copolyoxetane via cationic ring opening polymerization. The 1:1 copolymer composition and Mn (4 kDa) were confirmed by 1H NMR spectroscopy. After C–Br substitution by a series of tertiary amines, ionic liquid Cx-50 copolyoxetanes were obtained, where 50 is the mole percent of quaternary repeat units and “x” is quaternary alkyl chain length (2, 6, 8, 10, 12, 14, or 16 carbons). Modulated differential scanning calorimetry (MDSC) studies showed Tgs between −40 and −60 °C and melting endotherms for C14–50 and C16–50. Minimum inhibitory concentrations (MIC) were determined for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. A systematic dependence of MIC on alkyl chain length was found. The most effective antimicrobials were in the C6–50 to C12–50 range. C8–50 had better overall performance with MICs of 4 μg/mL, E. coli; 2 μg/mL, S. aureus; and 24 μg/mL, P. aeruginosa. At 5 × MIC, C8–50 effected >99% kill in 1 h against S. aureus, E. coli, and P. aeruginosa challenges of 108 cfu/mL; log reductions (1 h) were 7, 3, and 5, respectively. To provide additional insight into polycation interactions with bacterial membranes, a geometric model based on the dimensions of E. coli is described that provides an estimate of the maximum number of polycations that can chemisorb. Chain dimensions were estimated for polycation C8–50 with a molecular weight of 5 kDa. Considering the approximations for polycation chemisorption (PCC), it is surprising that a calculation based on geometric considerations gives a C8–50 concentration within a factor of 2 of the MIC, 4.0 (±1.2) μg/mL for E. coli. Cx-50 copolyoxetane cytotoxicity was low for human red blood cells, human dermal fibroblasts (HDF), and human foreskin fibroblasts (HFF). Selectivities for bacterial kill over cell lysis were among the highest ever reported for polycations indicating good prospects for biocompatibility.
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