Standardization of the Chloramine-T Method of Protein Iodination:
TL;DR: The method has been applied to the iodination of bovine serum albumin, ovalbumin, Salmonella flagella, and other proteins and the minimum amount of chloramine-T required for acceptable iodination is determined.
Abstract: SummaryA method is described for the controlled iodination of proteins. The potential, EMF, of the reaction mixture is monitored and used to determine the minimum amount of chloramine-T required for acceptable iodination. The method has been applied to the iodination of bovine serum albumin, ovalbumin, Salmonella flagella, and other proteins.
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TL;DR: The data suggest that intestinal epithelial cells may release LPS on chylomicrons from cell-associated pools, which may contribute to postprandial inflammatory responses or chronic diet-induced inflammation in chylomaticron target tissues.
556 citations
Cites methods from "Standardization of the Chloramine-T..."
...The LPS was labeled according to the chloramine-T protocol (28, 29)....
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TL;DR: Functional T cells were inferred from the ability of leaky mice to reject allogeneic skin grafts, a T cell-dependent reaction, and their lymphoid tissues retained the same general pattern of severe lymphocytic deficiency as scid mice.
Abstract: Although the majority of severe combined immune deficiency (scid) mice lack functional lymphocytes, some (2-23%) appear to develop a limited number of B and T cells between 3 and 9 mo old. Most of these leaky scid mice were shown to contain very few clones (less than or equal to 3) of Ig-producing plasmacytes. Clonal progeny were distributed unevenly in the lymphatic tissues and appeared as discrete plasmacytic foci. In many cases, individual clones persisted for several months and produced abnormally high concentrations of Ig that included multiple isotypes. Functional T cells were inferred from the ability of leaky mice to reject allogeneic skin grafts, a T cell-dependent reaction. Interestingly, approximately 40% of leaky mice developed thymic lymphomas. In other respects, leaky mice resembled regular scid mice; e.g., their splenic cells failed to express common lymphocyte antigens (Ly-5[B220], Ly-1) and to proliferate in response to lymphocyte mitogens. Histologically, their lymphoid tissues retained the same general pattern of severe lymphocytic deficiency as scid mice.
343 citations
TL;DR: It is suggested that CHIPS binds directly to the C5aR and FPR, thereby preventing the natural ligands from activating these receptors, making CHIPS a promising lead for the development of new anti-inflammatory compounds for diseases in which damage by neutrophils plays a key role.
Abstract: Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is an exoprotein produced by several strains of S. aureus, and a potent inhibitor of neutrophil and monocyte chemotaxis toward C5a and formylated peptides like fMLP. These chemoattractants act on their target cells by binding and activating the C5aR and formylated peptide receptor (FPR), respectively. In the present report, we examined the mechanism by which CHIPS affects both of these receptors. We showed that CHIPS blocked binding of anti-C5aR mAb and formylated peptide to human neutrophils as efficiently at temperatures of 0 and 37 degrees C, implying that it is independent of signal transducing systems. This was confirmed by showing that CHIPS acts completely independently of ATP. Additionally, CHIPS was not internalized upon binding to neutrophils. Furthermore, we showed that CHIPS binds specifically to the C5aR and FPR expressed on U937 cells. This binding was functional in blocking C5a- and fMLP-induced calcium mobilization in these cell lines. These results suggest that CHIPS binds directly to the C5aR and FPR, thereby preventing the natural ligands from activating these receptors. The apparent K(d) values of CHIPS for the C5aR and FPR were 1.1 +/- 0.2 nM and 35.4 +/- 7.7 nM, respectively. Moreover, after screening a wide variety of other G protein-coupled receptors, CHIPS was found to affect exclusively the C5aR and FPR. This selectivity and high-affinity binding with potent antagonistic effects makes CHIPS a promising lead for the development of new anti-inflammatory compounds for diseases in which damage by neutrophils plays a key role.
235 citations
Cites methods from "Standardization of the Chloramine-T..."
...CHIPS was iodinated using the chloramine-T method (12)....
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TL;DR: Data indicate that TLR4 modulation of PMN surface chemokine receptor expression subsequent to the downregulation of GRK2 and GRK5 expression is a critical determinant of PMn migration.
Abstract: Polymorphonuclear leukocytes (PMNs) are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites and killing pathogenic microbes. We addressed the role of chemokine receptor desensitization induced by G-protein-coupled receptor kinases (GRKs) in the feedback control of PMN migration. We show that the chemokine macrophage inflammatory protein-2 (MIP-2) induces GRK2 and GRK5 expression in PMNs through phosphoinositide-3-kinase (PI3K)-gamma signaling. We also show that lipopolysaccharide (LPS)-activated signaling through the Toll-like receptor (TLR)-4 pathway transcriptionally downregulates the expression of GRK2 and GRK5 in response to MIP-2. The reduced expression of GRKs lowers chemokine receptor desensitization and markedly augments the PMN migratory response. These data indicate that TLR4 modulation of PMN surface chemokine receptor expression subsequent to the downregulation of GRK2 and GRK5 expression is a critical determinant of PMN migration.
233 citations
TL;DR: The inhibitory effect of these 7 mAb has been investigated on a series of short‐term and long‐term expanded cloned CTL lines derived in vitro from an in vivo allosensitized individual exhibiting various specificities and appeared to be unrelated to their HLA‐A, B or C specificity or to their lytic activity.
Abstract: Monoclonal antibodies (mAb) derived from BALB/c mice immunized with human anti-HLA-A2 cloned cytotoxic T lymphocytes (CTL) were screened for their ability to block, in the absence of complement, the cytolytic activity of the immunizing CTL clone. Eight cytolysis-inhibiting mAb have been derived. One of these was directed against a monomorphic determinant expressed on HLA-class I molecules and thus probably inhibited cytolysis via a target cell antigen-masking effect. The 7 other mAb recognized "CTL function-associated structures" and did not have to interfere with target cell antigens in order to inhibit cytolysis. F(ab')2 and Fab fragments of these 7 mAb were also inhibitory. Competitive inhibition of binding and preliminary biochemical analysis suggested that these 7 mAb defined on cloned CTL various epitopes of a structure of 30 kDa disulfide-bonded into several multimeric forms when analyzed without reduction. The inhibitory effect of these 7 mAb has been investigated on a series of short-term (1 month) and long-term (greater than 10 months) expanded cloned CTL lines derived in vitro from an in vivo allosensitized individual exhibiting various specificities. Unexpectedly, only 10% of these CTL clones were inhibited. Flow cytofluorimetric analysis further revealed that noninhibited and inhibited CTL clones expressed similar amounts of the 30-kDa structure. Consequently, the inhibition of CTL was heterogeneous when analyzed at the clonal level and not simply related to the presence or absence of this structure. Furthermore, the ability of CTL clones to be inhibited appeared to be unrelated to their HLA-A, B or C specificity or to their lytic activity. The connections between this mAb-defined structure and CTL function are discussed.
187 citations
References
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01 Jan 1948
130 citations
"Standardization of the Chloramine-T..." refers background in this paper
...Equation ( 2 ) states that the potential, emf, determines the ratio of I 2 to I- (7)....
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35 citations
"Standardization of the Chloramine-T..." refers background in this paper
...The following constants facilitate calculation of the amounts of 1251-, KI, and protein required to give any desired specific activity and degree of substitution (9) : Carrier-free 1251has 2....
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