New syntheses of deuterated protoporphyrin-IX derivatives for heme protein nmr studies

An efficient total synthesis of 1,5-di(trideuteromethyl)protoporphyrin-IX (3) dimethyl ester from monopyrrole precursors is described, the synthesis proceeding through crystalline tripyrrene and a,c-biladiene salt intermediates. The 2- and 4-vinyl groups in (3) are formed from the corresponding (2-chloroethyl) substituents by way of base-promoted dehydrochlorination. In protio solvents, this synthetic step is shown to exchange out preferentially deuterons in the 1-methyl group, and this observation is exploited in an efficient synthesis of the 1,3-di(trideuteromethyl)protoporphyrin-IX (22) dimethyl ester from 2,4-diacetyldeuteroporphyrin-IX (20) dimethyl ester (which is in turn accessible from commercially available protoporphyrin-IX (5)). Thus, basic exchange in deuterated solvent of (20) gives the deuterated analog, which after reduction and dehydration gives the 1,3-di(trideuteromethyl)protoporphyrin-IX analog (22), in which the vinyl H₂ and propionic CH₂·CO functions have also become deuterated.     

Assessing wheat (Triticum aestivum L.) genetic diversity using quality traits, amplified fragment length polymorphisms, simple sequence repeats and proteome analysis

The genetic diversity among 10 Iranian bread wheat (Triticum aestivum) genotypes was analysed using 12 quality traits, 320 amplified fragment length polymorphisms (AFLP) polymorphic fragments, 491 simple sequence repeats (SSR) alleles and 294 proteome markers. The results revealed that the genotypes differed for quality traits, AFLP, SSR and proteome markers. The average genetic diversity based on quality traits (0.684 with a range of 0.266–0.997) was higher than AFLP (0.502 with a range of 0.328–0.717), SSR (0.503 with a range of 0.409–0.595) and proteome (0.464 with a range of 0.264–0.870) markers. Although there were apparent similarities between the groupings of particular genotypes, the overall correspondence between the distance matrices appeared to be rather low. In this study, the cluster analysis based on AFLP data showed the closest agreement with genotypes’ regions of origin or pedigree information. In addition to the genetic diversity assessment, specific proteins with known function were detected uniquely for the studied genotypes. Our results suggest that the classification based on quality traits and genotypic markers of these wheat genotypes will be useful for wheat breeders to plan crosses for positive traits.     

Activities of phosphomonoesterases as influenced by goethite and pyrite

Laboratory experiments were conducted to measure the activity of phosphomonoesterases in various zones of a geological outcrop. The outcrop contained an Entisol and partially weathered carbonaceous shales of Paleozoic age with differentiating zones rich in pyrite and goethite. Goethite is the most commonly occurring iron oxide in soils, and pyrite is often found in reconstructed soils of coal surface mines. The activity of acid phosphatase was significantly elevated in goethite‐rich zones compared to zones where pyrite was present. Alkaline phosphatase activity was reduced significantly in the presence of goethite but was not affected by pyrite. Experiments with pure pyrite and synthetic goethite substantiated the results obtained from the outcrop samples. The enhancing effect of goethite at lower pH values is probably due to chemisorption and removal of mineralized P, which increases the mineralization of organic P. At higher pH values goethite surfaces are negatively charged and repel phosphate anions. The exact mechanism for the inhibitory effect of pyrite on acid phosphatase is not known.     

Proteomics uncovers a role for redox in drought tolerance in wheat

Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in living systems. We applied a proteomic approach to study changes in wheat grain in response to drought, a major environmental parameter adversely affecting development and crop yield. Three wheat genotypes differing in genetic background were cultivated in field under well-watered and drought conditions by following a randomized complete block design with four replications. The overall effect of drought was highly significant as determined by grain yield and total dry matter. About 650 spots were reproducibly detected and analyzed on 2-DE gels. Of these, 121 proteins showed significant change under drought condition in at least one of the genotypes. Mass spectrometry analysis using MALDI-TOF/TOF led to the identification of 57 proteins. Two-thirds of identified proteins were thioredoxin (Trx) targets, in accordance with the link between drought and oxidative stress. Further, because of contrasting changes in the tolerant and susceptible genotypes studied, several proteins emerge as key participants in the drought response. In addition to providing new information on the response to water deprivation, the present study offers opportunities to pursue the breeding of wheat with enhanced drought tolerance using identified candidate genetic markers. The 2-DE database of wheat seed proteins is available for public access at http://www.proteome.ir.     

A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens

Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable monovalent co-engagement of a second target antigen in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.Key words: bispecific, mAb-Fv, Fc, heterodimer, CD16, CD3, HER2, HM1.24, anti-tumor, cancerDespite the enormous success of antibody-based therapeutics for the treatment of a variety of diseases, research efforts to improve their clinical efficacy continue. One avenue being explored is the engineering of new antigen binding sites to permit co-engagement of two distinct targets. Such engineered antibodies are commonly referred to as bispecifics, and a wide variety of formats have been described in references ¹ and ². Co-target antigens can include two targets believed to be causal in the pathology of a particular disease, e.g., two cytokines or growth factors.^3–5 Alternatively, the co-target pair may be a cell surface antigen and an immune receptor such that a novel “effector” mechanism can be built into the antibody, beyond those mediated naturally by the Fc region.²In the 1980s, bispecific antibodies were made by fusing two cell lines that each produced a single monoclonal antibody (mAb).⁶ Although the resulting hybrid hybridoma or quadroma did produce bispecifics, they were only a minor population and extensive purification was required to isolate the desired antibody. Antibody fragments provided an engineering solution to this problem; because they lack the complex quaternary structure of a full-length antibody, multiple variable regions can be linked in single genetic constructs. Antibody fragments of many different forms have been generated, including diabodies, single chain diabodies, tandem scFvs and F(ab'')₂ bispecifics.^2,7 While these formats can be expressed at high levels in bacteria and, arguably, may have benefits due to their small size, they suffer from poor half-life in vivo and can present manufacturing challenges related to their production and stability. For example, the rapid clearance of some fragment-based bispecifics requires that they be infused continuously via a portable pump over one to two months.⁸ The principal source of these limitations for fragment formats is the lack of an antibody Fc region with its associated structural and functional benefits, including large size that precludes renal filtration; high stability; binding to various Fc ligands, one of which maintains serum persistence (the neonatal Fc receptor FcRn) and binding to proteins A and G, which facilitates large scale purification.Recent work has attempted to address the shortcomings of fragment-based bispecifics by engineering a second antigen binding site into full-length antibody-like formats.^5,9–12 The presence of an Fc region in theory provides these formats with the developability and pharmacokinetic properties of standard IgG mAbs. However, because these constructs build new antigen binding sites on top of a homodimeric constant chain, binding to the new antigen is always bivalent. This consequence may pose a constraint depending on the co-targeting goal.For many immune receptors, cellular activation is accomplished by cross-linking of a monovalent binding interaction. The mechanism of cross-linking is typically mediated by antibody/antigen immune complexes, or via effector cell to target cell engagement. For example, the low affinity activating Fc gamma receptors (FcγRs) such as CD16 (FcγRIIIa) and CD32a (FcγRIIa) that mediate cellular killing bind monovalently to the antibody Fc region. While monovalent binding does not result in cellular signaling, upon effector cell engagement with the target cell, receptors are cross-linked and clustered on the cell surface, leading to activation.¹³ On T cells, CD3 activation occurs when its associated T-cell receptor (TCR) engages antigen-loaded major histocompatibility complex (MHC) on antigen-presenting cells in an avid cell-to-cell synapse.¹⁴ Bivalent antibodies targeting CD3 can elicit massive cytokine release, and the consequent toxicity has presented challenges for the development of anti-CD3 antibodies as drugs;^15,16 in contrast, monovalent binding of CD3 in Fab^17,18 and bispecific¹⁹ formats generates much lower levels of T-cell activation. For bispecifics, a consequence of this biology is that bivalent cross-linking of receptors can lead to non-specific activation of an effector cell in the absence of target cell.Thus, when the therapeutic goal is the co-engagement of an immune receptor, the desired binding may be monovalent rather than bivalent. This mode is incompatible with the majority of current full-length bispecifics. We describe an engineering solution to this problem that utilizes a heterodimeric Fc region to enable a single additional variable region to be built monomerically onto an antibody. Our new bispecific format, which we refer to as mAb-Fv, enables the simultaneous bivalent and monovalent co-engagement of distinct target antigens.     

Evaluating the effect of a mixture of two main conjugated linoleic acid isomers on hepatic steatosis in HepG2 cellular model

Molecular Biology Reports - Hepatic steatosis is an early form of non-alcoholic fatty liver disease (NAFLD), caused by abnormal fat deposition in the hepatocytes. Conjugated linoleic acid (CLA) is...     

Dominant-negative inhibitors of soluble TNF attenuate experimental arthritis without suppressing innate immunity to infection

TNF is a pleiotropic cytokine required for normal development and function of the immune system; however, TNF overexpression also induces inflammation and is associated with autoimmune diseases. TNF exists as both a soluble and a transmembrane protein. Genetic studies in mice have suggested that inflammation in disease models involves soluble TNF (solTNF) and that maintenance of innate immune function involves transmembrane TNF (tmTNF). These findings imply that selective pharmacologic inhibition of solTNF may be anti-inflammatory and yet preserve innate immunity to infection. To address this hypothesis, we now describe dominant-negative inhibitors of TNF (DN-TNFs) as a new class of biologics that selectively inhibits solTNF. DN-TNFs blocked solTNF activity in human and mouse cells, a human blood cytokine release assay, and two mouse arthritis models. In contrast, DN-TNFs neither inhibited the activity of human or mouse tmTNF nor suppressed innate immunity to Listeria infection in mice. These results establish DN-TNFs as the first selective inhibitors of solTNF, demonstrate that inflammation in mouse arthritis models is primarily driven by solTNF, and suggest that the maintenance of tmTNF activity may improve the therapeutic index of future anti-inflammatory agents.