Mechanistic Insight into the Enzymatic Reduction of Truncated Hemoglobin N of Mycobacterium tuberculosis: role of the CD loop and pre-A Motif in electron cycling

Background: The HbN of Mycobacterium tuberculosis carries a potent nitric-oxide dioxygenase activity despite lacking a reductase domain. Results: The NADH-ferredoxin reductase system acts as an efficient partner for the reduction of HbN. Conclusion: The interactions of HbN with the reductase are modulated by its CD loop and the Pre-A region. Significance: The present study provides new insights into the mechanism of electron transfer during nitric oxide detoxification by HbN.Fil: Singh, Sandeep. Institute of Microbial Technology; IndiaFil: Thakur, Naveen. Institute of Microbial Technology; IndiaFil: Oliveira, Ana. Universidad de Barcelona; EspañaFil: Petruk, Ariel Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Hade, Mangesh Dattu. Institute of Microbial Technology; IndiaFil: Sethi, Deepti. Institute of Microbial Technology; IndiaFil: Bidon Chanal, Axel. Universidad de Barcelona; EspañaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Datta, H.. Institute of Microbial Technology; IndiaFil: Parkesh, R.. Institute of Microbial Technology; IndiaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Luque, F. Javier. Universidad de Barcelona; EspañaFil: Dikshit, Kanak L.. Institute of Microbial Technology; Indi

Chemo-enzymatic synthesis and biological evaluation of photolabile nicotinic acid adenine dinuclotide phosphate (NAADP+).

A chemo-enzymatic synthesis of novel caged NAADP+ without the formation of multiple cage compounds has been achieved. The biological activity of the caged NAADP+ was demonstrated by its fast uncaging in intact sea-urchin eggs

NAADP-mediated channel 'chatter' in neurons of the rat medulla oblongata.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a potent Ca(2+)-mobilizing messenger that stimulates Ca(2+) release in a variety of cells. NAADP-sensitive Ca(2+) channels are thought to reside on acidic Ca(2+) stores and to be functionally coupled to IP(3) (inositol 1,4,5-trisphosphate) and/or ryanodine receptors located on the endoplasmic reticulum. Whether NAADP-sensitive Ca(2+) channels 'chatter' to other channels, however, is not clear. In the present study, we have used a cell-permeant NAADP analogue to probe NAADP-mediated responses in rat medulla oblongata neurons. NAADP-AM (NAADP-acetoxymethyl ester) evoked global cytosolic Ca(2+) signals in isolated neurons that were reduced in amplitude by removal of external Ca(2+), abolished by disruption of acidic compartments and substantially inhibited by blockade of ryanodine receptors. In rat medullary slices, NAADP-AM depolarized neurons from the nucleus ambiguus in the presence of intracellular EGTA, but not of the faster Ca(2+) chelator BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid]. Depolarization was also dependent upon extracellular Ca(2+), acidic stores and ryanodine receptors. In voltage-clamp mode, NAADP-AM induced an inward current with a reversal potential of approx. 0 mV. The results of the present study reveal the presence of acidic NAADP-sensitive Ca(2+) stores in medulla neurons, the mobilization of which results not only in global Ca(2+) signals but also in local signals that activate non-selective cation channels on the cell surface resulting in depolarization. Thus NAADP is capable of co-ordinating channels both within the cell interior and at the cell membrane representing a novel mechanism for excitation of central neurons

NAADP controls cross-talk between distinct Ca2+ stores in the heart.

In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load

Detecting microdamage in bone

Fatigue-induced microdamage in bone contributes to stress and fragility fractures and acts as a stimulus for bone remodelling. Detecting such microdamage is difficult as pre-existing microdamage sustained in vivo must be differentiated from artefactual damage incurred during specimen preparation. This was addressed by bulk staining specimens in alcohol-soluble basic fuchsin dye, but cutting and grinding them in an aqueous medium. Nonetheless, some artefactual cracks are partially stained and careful observation under transmitted light, or epifluorescence microscopy, is required. Fuchsin lodges in cracks, but is not site-specific. Cracks are discontinuities in the calcium-rich bone matrix and chelating agents, which bind calcium, can selectively label them. Oxytetracycline, alizarin complexone, calcein, calcein blue and xylenol orange all selectively bind microcracks and, as they fluoresce at different wavelengths and colours, can be used in sequence to label microcrack growth. New agents that only fluoresce when involved in a chelate are currently being developed – fluorescent photoinduced electron transfer (PET) sensors. Such agents enable microdamage to be quantified and crack growth to be measured and are useful histological tools in providing data for modelling the material behaviour of bone. However, a non-invasive method is needed to measure microdamage in patients. Micro-CT is being studied and initial work with iodine dyes linked to a chelating group has shown some promise. In the long term, it is hoped that repeated measurements can be made at critical sites and microdamage accumulation monitored. Quantification of microdamage, together with bone mass measurements, will help in predicting and preventing bone fracture failure in patients with osteoporosis

Synthesis and use of cell-permeant cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca(2+). cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca(2+). Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca(2+). Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions