Annexin A2 Mediates Apical Trafficking of Renal Na+-K+-2Cl? Cotransporter

The furosemide-sensitive Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca²⁺-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter.     

Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi‐compartment 3D perfusion bioreactors for extra‐corporeal liver support

Cultivation of primary human liver cells in innovative 3D perfusion multi‐compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale‐up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto‐ and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non‐parenchymal cells co‐cultivated in bioreactors were used for clinical extra‐corporeal liver support to bridge to liver transplantation. 3D tissue re‐structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular‐like structures, expression of HGFA and c‐Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto‐ and paracrine interactions between co‐cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment. Biotechnol. Bioeng. 2009;103: 817–827. © 2009 Wiley Periodicals, Inc.     

Deconstructing fragment-based inhibitor discovery

Fragment-based screens test multiple low-molecular weight molecules for binding to a target. Fragments often bind with low affinities but typically have better ligand efficiencies (DeltaG(bind)/heavy atom count) than traditional screening hits. This efficiency, combined with accompanying atomic-resolution structures, has made fragments popular starting points for drug discovery programs. Fragment-based design adopts a constructive strategy: affinity is enhanced either by cycles of functional-group addition or by joining two independent fragments together. The final inhibitor is expected to adopt the same geometry as the original fragment hit. Here we consider whether the inverse, deconstructive logic also applies--can one always parse a higher-affinity inhibitor into fragments that recapitulate the binding geometry of the larger molecule? Cocrystal structures of fragments deconstructed from a known beta-lactamase inhibitor suggest that this is not always the case.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Nitrocinnamate-functionalized gelatin: synthesis and "smart"hydrogel formation via photo-cross-linking

Gelatin having p-nitrocinnamate pendant groups (Gel-NC) was prepared via an efficient one-pot synthesis, yield >87%. (1)H NMR data indicated that 1 mol of gelatin was modified with 18 +/- 6 mol of the photosensitive group. Upon exposure to low-intensity 365 nm UV light and in the absence of photoinitiators or catalysts, Gel-NC cross-linked within minutes into a gelatin-based hydrogel as monitored by UV-vis spectroscopy. The degree of swelling of this biodegradable hydrogel in aqueous solutions responded to changes in Gel-NC concentration levels, the ionic strength of the aqueous solutions, and photo-cross-linking time. Topography changes associated with phase transition resulting from "photocleavage" of the hydrogel network with 254 nm UV light were studied with AFM. Both Gel-NC and its hydrogel expressed low toxicity to human neonatal fibroblast cells. In addition, gelatin-based microgels were prepared via the photo-cross-linking of Gel-NC within inverse micelles.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Red bone marrow dose estimation using several internal dosimetry models for prospective dosimetry-oriented radioiodine therapy

The aim of the present study was to review the available models developed for calculating red bone marrow dose in radioiodine therapy using clinical data. The study includes 18 patients (12 females and six males) with metastatic differentiated thyroid cancer. Radioiodine tracer of 73?±?16 MBq ¹³¹I was orally administered, followed by blood sampling (2 ml) and whole-body scans (WBSs) done at several time points (2, 6, 24, 48, 72, and ≥?96 h). Red bone marrow dose was estimated using the OLINDA/EXM 1.0, IDAC-Dose 2.1, and EANM models, the models developed by Shen and co-workers, Keizer and co-workers and Siegel and co-workers, and Traino and co-workers, as well as the single measurement model (SMM). The results were then compared to the standard reference model Revised Sgouros Model (RSM) reported by Wessels and co-workers. The mean dose deviations of the Traino, Siegel, Shen, Keizer, OLINDA/EXM, EANM, SMM, and IDAC-Dose 2.1 models from the RSM were ??17%, ??24%, 6%, ??29%, ??15%, 40%, 48%, and ??8%, respectively. The statistical analysis demonstrated no significant difference between the results obtained with the RSM and with those obtained with the Shen, Traino, OLINDA/EXM, and IDAC-Dose 2.1 models (t test; p_value > 0.05). However, a significant difference was found between RSM doses and those obtained with the EANM, SMM, and Keizer models (t test; p_value < 0.05). The correlation between red marrow dose from the SMM and EANM models was modest (R²?=?0.65), while the crossfire dose calculated with the OLINDA/EXM and IDAC-Dose 2.1 models were in good agreement with each other and with the reference model. The findings obtained indicate that most of the dosimetry models can be used for a reliable dosimetry, and the calculated total body doses can be considered as a reliable non-invasive option for a conservative activity planning. In addition, the excellent performance of the IDAC-Dose 2.1 model will be of particular importance for a practical and accurate dosimetry, with the advantages of allowing for the use of realistic advanced phantoms and updated dose fractions, and of providing information about the blood dose contribution to the red bone marrow.     

TDZ-specific plant regeneration in salad burnet

Various explants of salad burnet (Poterium sanguisorba L.=Sanguisorba minor Scop.) were cultured on semi-solid MS or B5 media containing factorial combinations of various plant growth regulators. Hypocotyl and petiole explants, after initial callus stage, regenerated prolific adventitious shoots via organogenesis, when placed on Murashige and Skoog basal medium containing 1–2 μM α-naphthaleneacetic acid and 4-20 μM thidiazuron. Any other growth regulator combination tested failed to respond. The addition of the biocide, Plant Preservative Mixture, was effective in controlling contamination and did not impair regeneration. Elongated shoots at 2–4 cm were transferred to rooting medium containing semi-solid Murashige and Skoog plus 1 μM α-naphthaleneacetic and rooted plants were transferred to the glasshouse. This is the first report on in vitro plant regeneration within the genusPoterium. This revised version was published online in June 2006 with corrections to the Cover Date.