Tryptophan residue is essential for immunoreactivity of a diagnostically relevant peptide epitope of A. fumigatus

The role of tryptophan (Trp¹⁷) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients’ IgG [K_D (equilibrium dissociation constant) = 2.74 e⁻⁸ ± 0.13 M] than the controls’ IgG (K_D = 2.97 e⁻⁶± 0.14 M), whereas chemically-modified P1 showed similar binding [K_D patients’ IgG = 3.25 e⁻⁷± 0.16 M, K_D controls’ IgG = 3.86 e⁻⁷± 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp¹⁷) is essential for immunoreactivity of P1.     

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.     

Interaction of resveratrol and genistein with nucleic acids

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.     

Discrimination in partnership: compartment-specific interactions of Hox proteins

Journal of Genetics -     

Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates

Polyphenols, coumarin (1,2-benzopyrone) and chromone (1,4-benzopyrone), are naturally occurring constituent of variety of plant species. They have attracted immense interest because of their diverse pharmacological activities. Not much was known about biological activities of acetyl derivative (polyphenolic acetates) of parent polyphenols. In previous investigations, we have conclusively established calreticulin transacetylase catalyzed activation of endothelial nitric oxide synthase (eNOS) by polyphenolic acetates. In the present work, calreticulin transacetylase of human peripheral blood mononuclear cells was characterized with respect to specificity for various polyphenolic acetates and its role in the activation of TNF-α induced nitric oxide synthase (iNOS). Peripheral blood mononuclear cells incubated with a model polyphenolic acetate, 7,8-diacetoxy-4-methylcoumarin (DAMC), along with l-arginine caused activation of NOS. The incubation of peripheral blood mononuclear cells with TNF-α and DAMC resulted in increased production of NO as compared to TNF-α alone. This increased NO production was attenuated by l-Nω-nitro-l-arginine methyl ester (l-NAME), a well known non-specific inhibitor of NOS, and 1400W (N-[3-(aminomethyl) benzyl] acetamidine), a specific inhibitor of human iNOS. These results substantiate the CRTAase catalyzed activation of iNOS. Further, expression of NOS isoforms by semi-quantitative PCR and real-time RT-PCR confirms the preponderance of iNOS in TNF-α treated peripheral blood mononuclear cells over the untreated one. It was also observed that polyphenolic acetates inhibit TNF-α mediated release of IL-6 from peripheral blood mononuclear cells.     

Interaction between Ataxin-2 Binding Protein 1 and Cubitus-interruptus during wing development in Drosophila

Animal growth and development is dependent on reiterative use of key signaling pathways such as Hedgehog (Hh) pathway. It is widely believed that Cubitus-interruptus (Ci) mediates all functions of Hh pathway. Here we report that CG32062, the Drosophila homologue of Ataxin-2 Binding Protein 1 (dA2BP1), functions as a cofactor of Ci to specify intervein region between L3 and L4 veins of the adult wing. Specifically, Ci-mediated transactivation of knot/collier (kn) in this region of the developing wing imaginal disc is dependent on dA2BP1 function. Protein interaction studies and chromatin-immunoprecipiation experiments suggest that Ci helps dA2BP1 to bind kn promoter, which in turn may help Ci to activate kn expression. These results suggest a mechanism by which Ci may activate targets such as kn, which do not have classical Ci/Gli-binding sites.     

Drosophila melanogaster Scramblases modulate synaptic transmission

Scramblases are a family of single-pass plasma membrane proteins, identified by their purported ability to scramble phospholipids across the two layers of plasma membrane isolated from platelets and red blood cells. However, their true in vivo role has yet to be elucidated. We report the generation and isolation of null mutants of two Scramblases identified in Drosophila melanogaster. We demonstrate that flies lacking either or both of these Scramblases are not compromised in vivo in processes requiring scrambling of phospholipids. Instead, we show that D. melanogaster lacking both Scramblases have more vesicles and display enhanced recruitment from a reserve pool of vesicles and increased neurotransmitter secretion at the larval neuromuscular synapses. These defects are corrected by the introduction of a genomic copy of the Scramb 1 gene. The lack of phenotypes related to failure of scrambling and the neurophysiological analysis lead us to propose that Scramblases play a modulatory role in the process of neurotransmission.     

Genotyping of alpha-thalassemia in microcytic hypochromic anemia patients from North India

Microcytic hypochromic anemia is a common condition in clinical practice and alpha-thalassemia has to be considered as a differential diagnosis. Molecular diagnosis of alpha-thalassemia is possible by polymerase chain reaction. The aim of this study was to evaluate the frequency of alpha-gene numbers in subjects with microcytosis. In total, 276 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] were studied. These include 125 with thalassemia trait, 48 with thalassemia major, 26 with sickle-cell thalassemia, 15 with E beta-thalassemia, 40 with iron-deficiency anemia, 8 with another hemolytic anemia, and 14 patients with no definite diagnosis. Genotyping for -alpha3.7 deletion, -alpha4.2 deletion, Hb Constant Spring, and a-triplications was done with polymerase chain reaction. The overall frequency of -alpha3.7 deletion in 276 individuals is 12.7%. The calculated allele frequency for a-thalassemia is 0.09. The subgroup analysis showed that co-inheritance of a-deletion is more frequent with the sickle-cell mutation than in other groups. We were able to diagnose 1/3 of unexplained cases of microcytosis as a-thalassemia carriers. The a-gene mutation is quite common in the Indian subcontinent. Molecular genotyping of a-thalassemia helps to diagnose unexplained microcytosis, and thus prevents unnecessary iron supplementation.