Antigen itself antibody: an alternative one‐step immunoassay for measuring the anti‐idiotypic antibody titer

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti‐idiotypic antibody. Our initial attempt to measure titers of mouse anti‐idiotypic antibody after idiotypic vaccination with HM‐1 killer toxin neutralizing monoclonal antibody (nmAb‐KT) failed. Because the injected antigen, nmAb‐KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen‐treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP‐conjugated‐nmAb‐KT used to measure the antibody titers in the antigen‐treated mice. Compared with control mice, signals were found in high anti‐nmAb‐KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non‐specific IgG. This method is amenable to long read lengths and will likely enable anti‐idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.     

Morpho-anatomical and physiological attributes for salt tolerance in sewan grass (Lasiurus scindicus Henr.) from Cholistan Desert,Pakistan

Three differently adapted populations of sewan grass (Lasiurus scindicus Henr.) were evaluated for structural and functional adaptations to high salinity. The habitats were Derawar Fort (DF, least saline, ECe 15.21), Bailahwala Dahar (BD, moderately saline, ECe 27.56 dS m^?1) and Ladam Sir (LS, highly saline, ECe 39.18 dS m^?1) from within the Cholistan Desert. The adaptive components of salt tolerance in sewan grass were assessed by determining various morpho–anatomical and physiological attributes. The degree of salt tolerance of all three ecotypes of L. scindicus from the saline habitats was compared in a controlled hydroponic system to evaluate the adaptive components that are expected to be genetically fixed during a long evolutionary process. Salinity tolerance in the most tolerant LS population relied on increased root length and total leaf area, restricted uptake of toxic Cl^?, increased uptake of Ca²⁺, high excretion of Na⁺, accumulation of organic osmolytes, high water use efficiency, increased root, thicker leaf and cortical region, intensive sclerification, large metaxylem vessels, and dense pubescence on abaxial leaf surface. The BD population (from moderately saline soil) relied on high Ca²⁺ uptake, Na⁺ excretion, epidermal thickness, large cortical cells, thick endodermis and large vascular tissue. The DF population (from less saline soil) showed a significant decrease in all morphological characteristics; however, it accumulated organic osmolytes for its survival under high salinities. Structural modifications in all three populations were crucial for checking undue water loss under physiological stress that is caused by high amounts of soluble salts in the soil.     

Implications of prion adaptation and evolution paradigm for human neurodegenerative diseases

There is a growing body of evidence indicating that number of human neurodegenerative diseases, including Alzheimer disease, Parkinson disease, fronto-temporal dementias, and amyotrophic lateral sclerosis, propagate in the brain via prion-like intercellular induction of protein misfolding. Prions cause lethal neurodegenerative diseases in humans, the most prevalent being sporadic Creutzfeldt-Jakob disease (sCJD); they self-replicate and spread by converting the cellular form of prion protein (PrP^C) to a misfolded pathogenic conformer (PrP^Sc). The extensive phenotypic heterogeneity of human prion diseases is determined by polymorphisms in the prion protein gene, and by prion strain-specific conformation of PrP^Sc. Remarkably, even though informative nucleic acid is absent, prions may undergo rapid adaptation and evolution in cloned cells and upon crossing the species barrier. In the course of our investigation of this process, we isolated distinct populations of PrP^Sc particles that frequently co-exist in sCJD. The human prion particles replicate independently and undergo competitive selection of those with lower initial conformational stability. Exposed to mutant substrate, the winning PrP^Sc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to the lowest stability. Thus, the evolution and adaptation of human prions is enabled by a dynamic collection of distinct populations of particles, whose evolution is governed by the selection of progressively less stable, faster replicating PrP^Sc conformers. This fundamental biological mechanism may explain the drug resistance that some prions gained after exposure to compounds targeting PrP^Sc. Whether the phenotypic heterogeneity of other neurodegenerative diseases caused by protein misfolding is determined by the spectrum of misfolded conformers (strains) remains to be established. However, the prospect that these conformers may evolve and adapt by a prion-like mechanism calls for the reevaluation of therapeutic strategies that target aggregates of misfolded proteins, and argues for new therapeutic approaches that will focus on prior pathogenetic steps.     

Mapping Spatially Resolved Charge Collection Probability within P3HT:PCBM Bulk Heterojunction Photovoltaics

Material properties in polymer and fullerene bulk heterojunctions (BHJs) such as donor to acceptor volume fraction, morphology, and molecular orientation critically influence light absorption, exciton dissociation, charge transport, and recombination, all of which are crucial device properties in organic photovoltaics (OPV). Spatial variation of BHJ properties normal to the substrate, caused by phase segregation, can thereby create corresponding spatial variations in the OPVs optoelectronic properties. Here, normally incident and wave‐guided optical modes are used to selectively excite localized regions within an inverted poly(3‐hexythiophene‐2,5‐diyl) and phenyl‐C61‐butyric acid methyl ester BHJ OPV and corresponding internal quantum efficiencies are measured to study the spatial‐dependent charge carrier collection probability within the BHJ. An electron‐limited charge collection profile is observed for a thick (920 nm) BHJ due to fullerene‐poor regions as a result of phase segregation. As the thickness of the BHJ is reduced (100 nm), charge transport is seen to be unaffected by the phase segregation. This has the potential to be a versatile non‐destructive characterization technique for measuring the spatially varying charge collection probability in thin film photovoltaics and will help enable optimum device design and characterization.     

Mechanism of Increased Tyrosine (Tyr<Superscript>99</Superscript>) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr⁹⁹ phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F_iO₂ of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr⁹⁹ and total) of calmodulin determined by Western blot using anti-phospho-(pTyr⁹⁹)-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr⁹⁹ calmodulin (ODxmm²) was 78.55 ± 10.76 in Nx, 165.05 ± 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 ± 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 ± 13.69 in Nx, 156.17 ± 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 ± 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr⁹⁹ phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr⁹⁹ phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr⁹⁹ phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr⁹⁹ phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.     

Theoretical design of bioinspired macromolecular electrets based on anthranilamide derivatives

Polypeptide helices possess considerable intrinsic dipole moments oriented along their axes. While for proline helices the dipoles originate solely from the ordered orientation of the amide bonds, for 3_10? and α‐helices the polarization resultant from the formation of hydrogen‐bond network further increases the magnitude of the macromolecular dipoles. The enormous electric‐field gradients, generated by the dipoles of α‐helices (which amount to about 5 D per residue with 0.15 nm residue increments along the helix), play a crucial role in the selectivity and the transport properties of ion channels. The demonstration of dipole‐induced rectification of vectorial charge transfer mediated by α‐helices has opened a range of possibilities for applications of these macromolecules in molecular and biomolecular electronics. These biopolymers, however, possess relatively large bandgaps. As an alternative, we examined a series of synthetic macromolecules, aromatic oligo‐ortho‐amides, which form extended structures with amide bonds in ordered orientation, supported by a hydrogen‐bond network. Unlike their biomolecular counterparts, the extended π‐conjugation of these macromolecules will produce bandgaps significantly smaller than the polypeptide bandgaps. Using ab initio density functional theory calculations, we modeled anthranilamide derivatives that are representative oligo‐ortho‐amide conjugates. Our calculations, indeed, showed intrinsic dipole moments oriented along the polymer axes and increasing with the increase in the length of the oligomers. Each anthranilamide residue contributed about 3 D to the vectorial macromolecular dipole. When we added electron donating (diethylamine) and electron withdrawing (nitro and trifluoromethyl) groups for n‐ and p‐doping, respectively, we observed that: (1) proper positioning of the electron donating and withdrawing groups further polarized the aromatic residues, increasing the intrinsic dipole to about 4.5 D per residue; and (2) extension of the π‐conjugation over some of the doping groups narrowed the band gaps with as much as 1 eV. The investigated bioinspired systems offer alternatives for the development of broad range of organic electronic materials with nonlinear properties. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10⁻³ M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.