Recombinant complement receptor 2 radiolabeled with [99mTc(CO)3]+: a potential new radiopharmaceutical for imaging activated complement

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [(99m)Tc(CO)(3)(OH(2))(3)](+)). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d(+) red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K(D)≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [(99m)Tc(CO)(3)(OH(2))(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d(+) RBCs. We conclude that rCR2-Tc(99m) has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.     

Ethephon mitigates nickel stress by modulating antioxidant system,glyoxalase system and proline metabolism in Indian mustard

The role of ethylene (through application of ethephon) in the regulation of nickel (Ni) stress tolerance was investigated in this study. Ethephon at concentration of 200 µl l^?1 was applied to mustard (Brassica juncea) plants grown without and with 200 mg kg^?1 soil Ni to study the increased growth traits, biochemical attributes, photosynthetic efficiency, nutrients content, activities of antioxidants such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase, glyoxalase systems and enhanced the proline metabolism. In the absence of ethephon, Ni increased oxidative stress with a concomitant decrease in photosynthesis, growth and nutrients content. However, application of ethephon positively increased growth traits, photosynthetic parameters, nutrients content and also elevated the generation of antioxidants enzymes and glyoxalase systems, proline production to combat oxidative stress. Plants water relations and cellular homeostasis were maintained through increased photosynthetic efficiency and proline production. This signifies the role of ethylene in mediating Ni tolerance via regulating proline production and photosynthetic capacity. Ethephon can be used as an exogenous supplement on plants to confer Ni tolerance. The results can be exploited to develop tolerance in plants via gene editing technology encoding enzymes responsible for proline synthesis, antioxidant defence, glyoxalase systems and photosynthetic effectiveness.

     

Cu(II) catalysed hydrolysis of thiaminepyrophosphate: structure-reactivity relationship

The kinetics of the Cu(II) catalysed hydrolysis of thiamine-pyrophosphate (TPP) has been studied in aqueous solution at 56,64 and 78° over a pH fange of 3.0 to 7.0 at a constant ionic strength of 0.10 M (KNO₃). The pH rate profiles were analysed and the overall rate constants resolved into individual specific rate constants relating to various Cu:TPP chelate species in solution. Activation parameters ΔH², ΔS^° and ΔG² for the specific rate constants of various chelate species of TPP are reported. The possible mechanism of the Cu(II) promoted hydrolysis of TPP is discussed. The structure-reactivity relationship is also discussed.     

PAS-mediated dimerization of soluble guanylyl cyclase revealed by signal transduction histidine kinase domain crystal structure

Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) because it is often associated with the heme-nitric oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 A resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCbeta1 H-NOXA homodimerization, heterodimerization of full-length heterodimeric sGC was mostly unaffected, likely due to the additional dimerization contacts of sGC in the coiled-coil and catalytic domains. Exceptions are mutations sGCalpha1 F285A and sGCbeta1 F217A, which each caused a drastic drop in NO stimulated activity, and mutations sGCalpha1 Q368A and sGCbeta1 Q309A, which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture.     

Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation

Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.     

Biosorption of copper by a bacterial biofilm on a flexible polyvinyl chloride conduit

Inexpensive technologies with less-than-optimal efficiencies as a strategy for countering economic restraints to pollution control have been evaluated by using a laboratory-scale biotreatment process for copper-containing effluent. Economizing measures include the use of polyvinyl chloride (PVC) cylinders fashioned from commercially available flexible PVC conduit to support a biofilm that was cultured in an inexpensive medium prepared in wastewater. The biofilm was challenged by aqueous copper solution in a bioreactor and subsequently analyzed under a scanning electron microscope with energy-dispersive X-ray microanalysis.     

Assessment of the serodiagnostic potential of nine novel proteins from Mycobacterium tuberculosis

To identify antigens that would improve the accuracy of serological diagnosis of active tuberculosis, we cloned the genes encoding nine potentially immunogenic secreted or surface-associated proteins of Mycobacterium tuberculosis. Recombinant proteins were reacted with sera from HIV-negative individuals with extrapulmonary tuberculosis (EP-TB) or HIV-positive individuals with pulmonary tuberculosis (TBH). Specific and high level antibody responses were obtained for four recombinant proteins, of which antigen GST-822 was recognized by 60% of EP-TB and 42% of TBH and antigen MBP-506 was recognized by 45% of EP-TB and 61% of TBH. These results suggest that these proteins are strong candidates as subunits in a polyvalent serodiagnostic test.     

Quantitation of the Calcium and Membrane Binding Properties of the C2 Domains of Dysferlin

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca²⁺ sensitive, the Ca²⁺ binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca²⁺ and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca²⁺ with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with K_d values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar K_d value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca²⁺ enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca²⁺ albeit with varying affinity and stoichiometry.     

Effect of peroxynitrite on human serum albumin: a multi technique approach

In this study, human serum albumin (HSA), the most abundant protein of blood plasma, was modified with varying concentrations of peroxynitrite. The peroxynitrite-induced changes in HSA was monitored by spectroscopy, SDS-PAGE, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal denaturation studies, and matrix-assisted laser desorption/inonization-time of flight mass spectrometry (MALDI-TOF MS). Aggregate formation was studied by thioflavin T binding and scanning electron microscopy (SEM). The results indicated formation of 3-nitrotyrosine, 6-nitrotryptophan, dityrosine, and carbonyls in modified samples and showed retarded mobility in SDS–polyacrylamide gel. Reduction in α-helicity and surface protein hydrophobicity confirmed the secondary and tertiary structure alterations in peroxynitrite-modified-HSA. Also, attachment of nitro group and increase in melting temperature was observed in modified sample. Furthermore, significant enhancement in the fluorescence intensity of ThT upon binding with peroxynitrite-modified-HSA and images under scanning electron microscope are suggestive of protein aggregation. It is, therefore, speculated that HSA modified by endogenously formed peroxynitrite might act as a trigger for nitration/aggregation and suggested the role of peroxynitrite-modified-HSA in SLE.     

A new species of Thrissina from Pakistan (Arabian Sea), with redescription of Thrissina whiteheadi (Wongratana 1983) (Clupeiformes: Engraulidae)

Ichthyological Research - Thrissina supra sp. nov. is described based on 15 specimens collected from Sindh, Pakistan. The new species is closely related to Thrissina whiteheadi (Wongratana 1983),...