Rajesh K. Grover

Ectopic B-cell clusters that infiltrate transplanted human kidneys are clonal

B cells and their immunoglobulin products participate in allograft rejection of transplanted human kidneys in which an interesting feature is the presence of a germinal center like B-cell clusters in the allograft. We report here that the immunoglobulin repertoires of these infiltrating B cells are highly restricted and the B cells within a cluster are clonal. Antibody libraries made from the infiltrating B cells of individual patients unexpectedly revealed that each patient utilizes a particular set of dominant germ line genes as well as dominant complementarity determining region 3. Comparison of kidney and peripheral blood from the same patient showed that the immunoglobulin genes from both compartments had dominant clones, but they differed. The lymphocytes that infiltrate the kidneys express the immunoglobulin gene somatic recombination machinery usually restricted to highly activated lymphocytes in germinal centers and lymphomas. An analogy can be made between the inescapable antigenic drive in chronic infection versus that in an allograft, both of which may lead to emergence of dominant B-cell clones and even lymphoid malignancy.

A Structurally Distinct Human Mycoplasma Protein that Generically Blocks Antigen-Antibody Union

Easy M Our immune systems can produce a vastly diverse repertoire of antibody molecules that each recognize and bind to a specific foreign antigen via a hypervariable region. However, there are a few bacterial antigens—such as Protein A, Protein G, and Protein L—that instead bind to the antibodys conserved regions and can bind to a large number of different antibodies. These high-affinity broad-spectrum antibody-binding properties have been widely exploited both in the laboratory and in industry for purifying, immobilizing, and detecting antibodies. Grover et al. (p. 656) have now identified Protein M found on the surface of human mycoplasma, which displays even broader antibody-binding specificity. The crystal structure of Protein M revealed how Protein-M binding blocks the antibodys antigen binding site. This mechanism may be exploited by mycoplasma to escape the humoral immune response. High-affinity binding of Protein M to a very broad range of human antibodies may find widespread immunochemical applications. We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.

Cholesterol Secosterol Aldehydes Induce Amyloidogenesis and Dysfunction of Wild Type Tumor Protein p53

Epidemiologic and clinical evidence points to an increased risk for cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and -B (ALD), but not the polyunsaturated fatty acid (PUFA)-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by the analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction and cancer risk.

The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1

The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (β-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a ‘helical bouquet’ with a ‘ribbon’ helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.

A novel diterpenoid lactone-based scaffold for the generation of combinatorial libraries †

Abstract A novel labdane diterpenoid-based scaffold: 14-deoxyandrographolide has been identified for the generation of combinatorial libraries using solid-phase methods. To allow access to a larger pool of building blocks with wide structural diversity, 14-deoxy andrographolide has been linked to 2-chlorotrityl chloride resin. This was followed by a series of solid-phase reactions carried out on the C-3 hydroxyl and the C-8(17) double bond. The details of our synthetic strategies involving coupling of the resin, solid-phase acetylation under neutral conditions, esterification, oxidation, epoxidation reactions and formation of oxime esters from oximes are presented here. The utility of the scaffold has been demonstrated by synthesizing a small, 20 member, library of 14-deoxyandrographolide derivatives via esterification and oxime ester formation using five alkyl and five aryl carboxylic acids.

The costimulatory immunogen LPS induces the B-Cell clones that infiltrate transplanted human kidneys

The mechanism of chronic rejection of transplanted human kidneys is unknown. An understanding of this process is important because, chronic rejection ultimately leads to loss of the kidney allograft in most transplants. One feature of chronic rejection is the infiltration of ectopic B-cell clusters that are clonal into the transplanted kidney. We now show that the antibodies produced by these B-cells react strongly with the core carbohydrate region of LPS. Since LPS is a costimulatory immunogen that can react with both the B-cell receptor (BCR) and the Toll-like receptor 4 (TLR4), these results suggest a mechanism for the selective pressure that leads to clonality of these B-cell clusters and opens the possibility that infection and the attendant exposure to LPS plays a role in the chronic rejection of human kidney transplants. If confirmed by clinical studies, these results suggest that treating patients with signs of chronic rejection with antibiotics may improve kidney allograft survival.

Solid phase synthesis of 3,5-disubstituted oxazolidin-2-ones

Abstract A versatile method for the solid phase synthesis of oxazolidin-2-ones is described. A resin bound phenolic group was treated with (±)-epichlorohydrin followed by opening of the epoxide ring with sodium azide. The resulting 1-azido-3-aryloxypropan-2-ol was treated with p -nitrophenylchloroformate and subsequent Staudingers cyclization using PPh 3 yielded a 5-substituted oxazolidinone. Finally, additional diversity at position 3 was introduced by treating the 5-substituted oxazolidinone with an alkyl halide in the presence of NaH to give the desired compound in high yield and purity.

Antibodies from combinatorial libraries use functional receptor pleiotropism to regulate cell fates.

To date, most antibodies from combinatorial libraries have been selected purely on the basis of binding. However, new methods now allow selection on the basis of function in animal cells. These selected agonist antibodies have given new insights into the important problem of signal transduction. Remarkably, when some antibodies bind to a given receptor they induce a cell fate that is different than that induced by the natural agonist to the same receptor. The fact that receptors can be functionally pleiotropic may yield new insights into the important problem of signal transduction.