No apparent cost of evolved immune response in Drosophila melanogaster

Maintenance and deployment of the immune system are costly and are hence predicted to trade‐off with other resource‐demanding traits, such as reproduction. We subjected this longstanding idea to test using laboratory experimental evolution approach. In the present study, replicate populations of Drosophila melanogaster were subjected to three selection regimes—I (Infection with Pseudomonas entomophila), S (Sham‐infection with MgSO₄), and U (Unhandled Control). After 30 generations of selection flies from the I regime had evolved better survivorship upon infection with P. entomophila compared to flies from U and S regimes. However, contrary to expectations and previous reports, we did not find any evidence of trade‐offs between immunity and other life history related traits, such as longevity, fecundity, egg hatchability, or development time. After 45 generations of selection, the selection was relaxed for a set of populations. Even after 15 generations, the postinfection survivorship of populations under relaxed selection regime did not decline. We speculate that either there is a negligible cost to the evolved immune response or that trade‐offs occur on traits such as reproductive behavior or other immune mechanisms that we have not investigated in this study. Our research suggests that at least under certain conditions, life‐history trade‐offs might play little role in maintaining variation in immunity.     

Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.     

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra‐physiological levels of phospho‐tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry‐based phospho‐proteomics, we show that glucose withdrawal initiates a unique signature of phospho‐tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal‐induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS‐mediated cell death. Taken together, these findings illustrate the systems‐level cross‐talk between metabolism and signaling in the maintenance of cancer cell homeostasis.     

Differential specificities and simultaneous occupancy of human MutSalpha nucleotide binding sites

We have examined the permissible nucleotide occupancy states of human MutSalpha. The MSH2.MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5'-O-(thiotriphosphate) (ATPgammaS), with a K(d) for each nucleotide of about 1 microm. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutSalpha heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPgammaS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutSalpha is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutSalpha-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutSalpha.heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutSalpha and ADP or a triphosphate analogue. These findings imply that MutSalpha has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP.MutSalpha.ATP ternary complex has an important role in mismatch repair.     

DNA interaction studies of ethylenediaminetetraaceticacid food additive and selenium effect in DNA cleavage-inhibition

The interaction of native calf thymus DNA with ethylenediaminetetraaceticacid (EDTA) in Tris-HCl buffer at pH?=?7.8 (at this pH EDTA forms a disodium salt) was investigated. EDTA is widely used in medicine, food technology, and the chemical industry. The DNA binding mode of EDTA was monitored by absorption spectrophotometry, circular dichrosim, viscometry, and gel electrophoresis. Ultraviolet spectra of DNA showed small hyperchromicity with increase in EDTA concentration. The circular dichrosim signals at 245 and 280?nm indicated structural changes in DNA structure, and no significant effect on DNA viscosity was observed in the presence of increasing amounts of EDTA. Results are indicative of an outside, nonintercalative binding mode of EDTA to DNA. Moreover, gel electrophoresis studies showed considerable oxidative cleavage of plasmid DNA by EDTA. Mechanistic aspects of the chemical nuclease reactions were investigated using the OH radical quencher dimethylsulfoxide. In the presence of selenium, DNA cleavage by EDTA was inhibited.     

DNA interaction studies of sesamol (3,4-methylenedioxyphenol) food additive

The interaction of native calf thymus DNA (CT-DNA) with sesamol (3,4-methylenedioxyphenol) in Tris–HCl buffer at neutral pH 7.4 was monitored by absorption spectrophotometry, viscometry and spectrofluorometry. It is found that sesamol molecules could interact with DNA outside and/or groove binding modes, as are evidenced by: hyperchromism in UV absorption band, very slow decrease in specific viscosity of DNA, and small increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of sesamol, which indicates that it is able to partially release the bound MB. Furthermore, the enthalpy and entropy of the reaction between sesamol and CT-DNA showed that the reaction is enthalpy-favored and entropy-disfavored (ΔH = ?174.08 kJ mol^?1; ΔS = ?532.92 J mol^?1 K^?1). The binding constant was determined using absorption measurement and found to be 2.7 × 10⁴ M^?1; its magnitude suggests that sesamol interacts to DNA with a high affinity.     

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (K_i = 0.120 ± 0.005 nm) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t_½ ∼12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE.