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1.
A noncytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the cytolysin/perforin gene 总被引:5,自引:0,他引:5
To test the granule exocytosis model for lymphocyte cytotoxicity, we have expressed the gene for the cytotoxic lymphocyte granule protein cytolysin (perforin) in the noncytotoxic rat basophilic leukemia (RBL) cell line, which undergoes granule exocytosis when its high affinity IgE receptor is cross-linked. Homogenates of RBL-cytolysin (RBL-cy) transfectants showed a calcium-dependent hemolytic activity in dense granule fractions, demonstrating that the expressed cytolysin protein was correctly targeted to secretory granules. RBL-cy transfectants showed a potent and calcium-dependent cytolytic activity against IgE-coated RBCs, while the parental RBL line was not cytotoxic under these conditions. RBL-cy cells did not lyse non-IgE-coated RBCs copelleted with targets: this sparing of "innocent bystanders" parallels cytotoxic T lymphocyte lysis and suggests a polarized secretion of cytolysin. In contrast to RBC targets, IgE-coated tumor cells were much less sensitive to lysis by RBL-cy transfectants. 相似文献
2.
Site-directed mutagenesis of the charged residues near the carboxy terminus of the colicin E1 ion channel 总被引:3,自引:0,他引:3
Colicin E1 was altered by oligonucleotide-directed mutagenesis at the site of three charged residues on the COOH side of the 35-residue hydrophobic segment in the channel-forming domain. Asp-509 is one of five conserved acidic residues in the channel domain of colicins A, B, E1, Ia, and Ib and is the first charged residue following the hydrophobic segment, followed by the basic residues Lys-510 and Lys-512. Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon. Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a nonsuppressing cell strain harboring the mutant plasmid while full-length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gln, and Lys-512 to Tyr, were obtained by using appropriate suppressor strains. The truncated colicins displayed (i) a low cytotoxicity, approximately 1% of intact wild-type colicin, (ii) 10-fold less in vitro channel activity with liposomes, and (iii) reduced labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer. (iv) The truncated mutants also displayed an altered conformation at pH 6 that allowed greater binding and activity with liposomes at this pH relative to wild type. The cytotoxicity of single residue substitutions at Asp-509 showed a range of cytotoxicities, wild type greater than Ser-509 greater than Gln-509 greater than Leu-509, although none of these changes greatly affected the in vitro channel activity or pH dependence.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
The ATP-binding-cassette transmembrane transporters (ABC transporters)
known from vertebrates belong to four major subfamilies: (1) the P-
glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance
regulators (CFTR); (3) the Tap proteins encoded with the major
histocompatibility complex of mammals; and (4) the peroxisomal membrane
proteins. Both Pgp and CFTR have a structure suggesting a past internal
gene duplication; a phylogenetic analysis indicated that these duplications
occurred independently, while an independent tandem gene duplication
occurred in the case of the Tap family. Both the Pgp and Tap proteins show
evidence of relationship to bacterial ABC transporters lacking internal
duplication, and both are significantly more closely related to the HlyB
and MsbA families of transporters from purple bacteria than they are to ABC
transporters from nonpurple bacteria. The simplest hypothesis to explain
this observation is that eukaryotic Pgp and Tap genes are descended from a
mitochondrial gene or genes that were subsequently translocated to the
nuclear genome. The Pgp genes of eukaryotes are characterized by a
remarkable degree of convergent evolution between the ATP-binding cassettes
of their N- terminal and C-terminal halves, whereas no such convergence is
seen between the two halves of CFTR genes or between the duplicated Tap
genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain
not found in other ABC transporters apart from CFTR, showed high levels of
both synonymous and nonsynonymous difference in comparisons among different
mammalian species, suggesting that this region is a mutational hot spot.
相似文献
4.
5.
X Xiao G Hintermann AL Demanin J Piret 《Journal of industrial microbiology & biotechnology》1996,16(4):261-262
Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis. 相似文献
6.
Formation of ion-conducting channels by the membrane attack complex proteins of complement. 下载免费PDF全文
The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different. 相似文献
7.
ERIC S. BONO PATRICK SMOLINSKI AL CASAGRANDA JUNDE XU 《Computer methods in biomechanics and biomedical engineering》2013,16(2):125-131
The Shear-slip Mesh Update Method (SSMUM) is being used in flow simulations involving large but regular displacements of one or more boundaries of the computational domain. We follow up the earlier discussion of the method with notes on practical implementation aspects. In order to establish a benchmark problem for this class of flow problems, we define and report results from a two-dimensional viscous flow around a rotating stirrer in a square chamber. The application potential of the method is demonstrated in the context of biomedical design problem, as we perform an analysis of blood flow in a centrifugal left ventricular assist device, or blood pump, which involves a rotating impeller in a non-axisymmetric housing. 相似文献
8.
9.
HENRY SILVERMAN BABIKER AHMED SAMAR AJEILET SUMAIA AL‐FADIL SUHAIL AL‐AMAD HADIR EL‐DESSOUKY IBRAHIM EL‐GENDY MOHAMED EL‐GUINDI MUSTAFA EL‐NIMEIRI RANA MUZAFFAR AZZA SALEH 《Developing world bioethics》2010,10(2):70-77
To help ensure the ethical conduct of research, many have recommended educational efforts in research ethics to investigators and members of research ethics committees (RECs). One type of education activity involves multi‐day workshops in research ethics. To be effective, such workshops should contain the appropriate content and teaching techniques geared towards the learning styles of the targeted audiences. To ensure consistency in content and quality, we describe the development of a curriculum guide, core competencies and associated learning objectives and activities to help educators organize research ethics workshops in their respective institutions. The curriculum guide is divided into modular units to enable planners to develop workshops of different lengths and choose content materials that match the needs, abilities, and prior experiences of the target audiences. The content material in the curriculum guide is relevant for audiences in the Middle East, because individuals from the Middle East who participated in a Certificate Program in research ethics selected and developed the training materials (e.g., articles, powerpoint slides, case studies, protocols). Also, many of the activities incorporate active‐learning methods, consisting of group work activities analyzing case studies and reviewing protocols. The development of such a workshop training curriculum guide represents a sustainable educational resource to enhance research ethics capacity in the Middle East. 相似文献
10.
McGaughey GB Citron M Danzeisen RC Freidinger RM Garsky VM Hurni WM Joyce JG Liang X Miller M Shiver J Bogusky MJ 《Biochemistry》2003,42(11):3214-3223
The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response. 相似文献