Model-Guided Mutagenesis Drives Functional Studies of Human NHA2, Implicated in Hypertension

Human NHA2 is a poorly characterized Na⁺/H⁺ antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity, and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by an extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well-known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.     

Binuclear manganese(III) complexes of an unsymmetric pyrazolate-based compartmental ligand with hard donor set

The synthesis, crystal structure and magnetic properties of manganese(III) binuclear complexes [Mn^III₂(L-3Н)₂(CH₃ОH)₄]·2CH₃ОH (1) and [Mn^III₂(L-3Н)₂(Py)₄]·2Py (2) (L = 3-[(1E)-N-hydroxyethanimidoyl]-4-methyl-1H-pyrazole-5-carboxylic acid) are reported. The ligand contains two distinct donor compartments formed by the pyrazolate-N and the oxime or the carboxylic groups. The complexes were characterized by X-ray single crystal diffraction, revealing that both 1 and 2 consist of dinuclear units in which the two metal ions are linked by double pyrazolate bridges with a planar {Mn₂N₄} core. Cryomagnetic measurements show antiferromagnetic interaction with g = 1.99, J = −3.6 cm⁻¹, Θ = −2.02 K for 1 and g = 2.00, J = −3.7 cm⁻¹, Θ = 1.43 K for 2.     

Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl₂ (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl₂ (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl₂ solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.     

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.     

Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling

Protein kinase C (PKC) family members transduce an abundance of diverse intracellular signals. Here we address the role of spatial and temporal segregation in signal specificity by measuring the activity of endogenous PKC at defined intracellular locations in real time in live cells. We targeted a genetically encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter (CKAR) (Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) J. Cell Biol. 161, 899-909), to the plasma membrane, Golgi, cytosol, mitochondria, or nucleus by fusing appropriate targeting sequences to the NH2 or COOH terminus of CKAR. Measuring the phosphorylation of the reporter in the presence of PKC inhibitors, activators, and/or phosphatase inhibitors shows that activity at each region is under differential control by phosphatase activity; nuclear activity is completely suppressed by phosphatases, whereas membrane-associated activity is the least suppressed by phosphatases. UTP stimulation of endogenous P2Y receptors in COS 7 cells reveals spatiotemporally divergent PKC responses. Imaging the second messengers Ca2+ and diacylglycerol (DAG) reveal that PKC activity at each location is driven by an initial spike in Ca2+, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.     

Rapid test for distinguishing membrane-active antibacterial agents

In the search for antibacterial agents with a novel mode-of-action (MOA) many targeted cellular and cell-free assays are developed and used to screen chemical and natural product libraries. Frequently, hits identified by the primary screens include compounds with nonspecific activities that can affect the integrity and function of bacterial membrane. For a rapid dereplication of membrane-active compounds, a simple method was established using a commercially available Live/Dead(R) Bacterial Viability Kit. This method utilized two fluorescent nucleic acid stains, SYTO9 (stains all cells green) and propidium iodide (stains cells with damaged membrane red) for the drug-treated bacterial cells. The cells were then either examined visually by fluorescence microscopy or their fluorescence emissions were recorded using a multi-label plate reader set to measure emissions at two different wavelengths. The ratio of green versus red was compared to a standard curve indicating the percentage of live versus dead bacteria. Nine known antibiotics and 14 lead compounds from various antibacterial screens were tested with results consistent with their MOA.     

Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms

The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor.