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Biomedical subjects

D Shevchenko

Publications and source records attributed to D Shevchenko.

6 recordsLinked to original sources
PubMed2000

Endotoxin-mimetic effect of antibodies against Toll-like receptor 4.

Monospecific, affinity-purified polyclonal antibodies reacting with the amino-terminal half of the mouse Toll-like receptor 4 (Tlr4) ectodomain failed to block LPS effects and, to the contrary, were capable of inducing TNF synthesis when applied to mouse macrophages and cross-linked with a secondary antibody. This effect was observed with macrophages derived from C3H/HeN and C57BL/10ScSn mice, but not with macrophages derived from C3H/HeJ or C57BL/10ScCr mice, indicating a specific, Tlr4-dependent effect. Neither primary nor secondary antibody caused any response if administered in the absence of the other reagent, nor was any response observed in cells from mice lacking Tlr4, or bearing the Lps(d) mutation of Tlr4. These findings support several conclusions. Tlr4, the essential transducer of LPS responses, may act independently of LPS itself. LPS needs not be internalized, nor must it bind to a secondary target within the cell in order to exert its effect; rather, the receptor alone is required for initiation of a signal. The data are consistent with the hypothesis that a conformational change in Tlr4 is required for activation via this receptor, and reveal that the amino-terminal half of the Tlr4 ectodomain is a target sufficient for antibody-mediated activation.

Animals↗
PubMed1999

Physicochemical evidence that Treponema pallidum TroA is a zinc-containing metalloprotein that lacks porin-like structure.

Although TroA (Tromp1) was initially reported to be a Treponema pallidum outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein's amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive beta-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins.

Bacterial Proteins↗
PubMed1997

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function.

Azirines↗
PubMed1995

Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue.

Protein export signals from the low-passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence. One PhoA+ clone (BbK2.10-PhoA) was derived from a borrelial lipoprotein. Although the polypeptide encoded by the full-length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B. burgdorferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross-reactive. The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes. Southern blot analysis revealed that bbk2.10 is a single-copy gene and that the B. burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10. It was particularly noteworthy that ospF, but not bbk2.10, was expressed in vitro while B. burgdorferi-infected mice generated antibodies reactive with both lipoproteins. To help confirm that the BbK2.10-reactive antibodies produced by the B. burgdorferi-infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10-specific antisera. Taken together, these data demonstrate that BbK2.10, BbK2.11, and OspF comprise a B. burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection.

Amino Acid Sequence↗