Integral membrane proteins of Treponema pallidum subsp. pallidum (T. pallidum) were identified by phase partitioning with the nonionic detergent Triton X-114; antigens with apparent molecular masses of 47,38,36,34,32,17, and 15 kilodaltons (kDa) were identified in the detergent phase. Immunoblotting with murine monoclonal antibodies directed against pathogen-specific 47and 34-kDa T. pallidum antigens confirmed their presence in the detergent phase. Endoflagellar proteins of T. pallidum were not detected in immunoblots of detergent-phase proteins when monospecific antisera directed against endoflagelia of the nonpathogenic T. phagedenis biotype Reiter were used. At detergent concentrations (0.02 and 0.1%) which appeared to solubilize selectively the outer membranes of treponemes radiolabeled with 355 in vitro, limited amounts of detergentphase proteins were immunoprecipitated. Greater amounts of detergent-phase proteins were extracted at higher detergent concentrations (0.5 and 2.0%) which resulted in both outer membrane solubilization and ultrastructural derangements of the residual cytoplasmic bodies. Furthermore, Triton X-114 extraction of both intact treponemes and organisms without outer membranes yielded detergent phases with similar protein profiles. The results of these experiments indicate that the hydrophobic proteins identified by Triton X-114 are not located exclusively in the T. pallidum outer membrane. The results are also consistent with the hypothesis that the T. pallidum outer membrane is a protein-deficient lipid bilayer. 490 on July 11, 2020 by guest
A flagellar adhesion-induced signal sent during the mating reaction of the biflagellate alga, Chlamydomonas reinhardtii, initiates release of cell-wall-degrading enzymes, activation of mating structures, and cell fusion. The nature of this signal is unknown, but it may be mediated by an adhesion-induced change (activation) of flagellar tips. The studies reported here show that lidocaine, a local anesthetic that is reported to interfere with the movement of divalent cations across cell membranes, reversibly blocks cell wall loss and gametic fusion without blocking adhesion or flagellar tip activation. In these experiments lidocaine inhibited both the initial rates and the extent of wall loss and zygote formation. Studies with gametes of a paralyzed flagellar mutant, pf 17, revealed that lidocaine also blocked flagellar surface motility (visualized as movement of polystyrene beads) at concentrations of the inhibitor which also prevented gametic fusion. The concentration of lidocaine required to block cell fusion was dependent on the concentration of calcium or magnesium in the medium. In the absence of added calcium, 0.5 mM lidocaine inhibited fusion by 70%. In 0.5 mM calcium, 0.5 mM lidocaine had no effect on fusion and 2 mM lidocaine was required for 90% inhibition. The results suggest that divalent cations may play a critical role in sexual signalling in Chlamydomonas.
Sequencing techniques for singleand double-stranded DNA were used to determine the nucleotide sequence of the gene encoding P2, the major outer membrane (porin) protein of Haemophilus influenzae type b (Hib).The open reading frame encoding the P2 protein comprised 361 amino acid codons. Comparison of the inferred amino acid sequence with data obtained by amino acid sequencing of the N terminus of the mature or fully processed P2 protein revealed that this protein has a signal peptide composed of 20 amino acids. N-terminal amino acid sequencing of tryptic peptides derived from purified P2 allowed direct identification of 158 of the 341 amino acids in the fully processed P2 protein; there was 100% correlation between these amino acid sequences and that inferred from the nucleotide sequence. The amino acid sequence of Hib P2 protein had 23 to 25% homology with the sequence of the OmpF porin of Escherichia coli and with that of the Neisseria gonorrhoeae porin P.IA. Codon usage in the Hib P2 gene was significantly different from that observed for a gene encoding a porin of E. coli. DNA hybridization studies indicated that there is a single copy of the P2 gene in the Hib chromosome. The availability of the nucleotide and amino acid sequences for the Hib P2 protein will facilitate investigation of the antigenic characteristics and structure-function relationship of this porin. 1100 on August 1, 2020 by guest
HIV-1 replicates preferentially in IL-4 producing CD4 T cells for unclear reasons. We show increased HIV-1 expression is irrespective of viral tropism for chemokine receptors as previously suggested, but rather transcription of the HIV-1 long terminal repeat (LTR) is increased in IL-4 producing CD4 T cells. Increased expression of HIV-1 message is also confirmed in IL-4 producing CD4 T cells from HIV-1 infected individuals ex vivo. In exploring a transcriptional mechanism, we identify a novel c-maf (required for IL-4 expression) transcription factor binding site just upstream of the dual NFκB/NFAT binding sites in the proximal HIV-1 LTR. We demonstrate c-maf binds this site in vivo and synergistically augments HIV-1 transcription in cooperation with NFAT2 and NFκB p65, but not NFAT1 nor NFκB p50. Conversely, siRNA inhibition of c-maf reduces HIV-1 transcription in IL-4 producing T cells. Thus, c-maf increases HIV-1 expression in IL-4 producing CD4 T cells by binding the proximal HIV-1 LTR and augmenting HIV-1 transcription in partnership with NFAT2 and NFκB p65, specifically. This has important implications for selective targeting of transcription factors during HIV-1 infection since, over the course of HIV-1 progression/AIDS, IL-4 producing T cells frequently predominate and substantially contribute to disease pathology.
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