The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Biomimetic coating of organic polymers with a protein-functionalized layer of calcium phosphate: the surface properties of the carrier influence neither the coating characteristics nor the incorporation mechanism or release kinetics of the protein

Polymers that are used in clinical practice as bone-defect-filling materials possess many essential qualities, such as moldability, mechanical strength and biodegradability, but they are neither osteoconductive nor osteoinductive. Osteoconductivity can be conferred by coating the material with a layer of calcium phosphate, which can be rendered osteoinductive by functionalizing it with an osteogenic agent. We wished to ascertain whether the morphological and physicochemical characteristics of unfunctionalized and bovine-serum-albumin (BSA)-functionalized calcium-phosphate coatings were influenced by the surface properties of polymeric carriers. The release kinetics of the protein were also investigated. Two sponge-like materials (Helistat® and Polyactive®) and two fibrous ones (Ethisorb and poly[lactic-co-glycolic acid]) were tested. The coating characteristics were evaluated using state-of-the-art methodologies. The release kinetics of BSA were monitored spectrophotometrically. The characteristics of the amorphous and the crystalline phases of the coatings were not influenced by either the surface chemistry or the surface geometry of the underlying polymer. The mechanism whereby BSA was incorporated into the crystalline layer and the rate of release of the truly incorporated depot were likewise unaffected by the nature of the polymeric carrier. Our biomimetic coating technique could be applied to either spongy or fibrous bone-defect-filling organic polymers, with a view to rendering them osteoconductive and osteoinductive.

Expression, localization, and functional model of cholesterol transporters in lactating and nonlactating mammary tissues of murine, bovine, and human origin

Members of the ATP-binding cassette (ABC) transporters play a pivotal role in cellular lipid efflux. To identify candidate cholesterol transporters implicated in lipid homeostasis and mammary gland (MG) physiology, we compared expression and localization of ABCA1, ABCG1, and ABCA7 and their regulatory genes in mammary tissues of different species during the pregnancy-lactation cycle. Murine and bovine mammary glands (MGs) were investigated during different functional stages. The abundance of mRNAs was determined by quantitative RT-PCR. Furthermore, transporter proteins were localized in murine, bovine, and human MGs by immunohistochemistry. In the murine MG, ABCA1 mRNA abundance was elevated during nonlactating compared with lactating stages, whereas ABCA7 and ABCA1 mRNA profiles were not altered. In the bovine MG, ABCA1, ABCG1, and ABCA7 mRNAs abundances were increased during nonlactating stages compared with lactation. Furthermore, associations between mRNA levels of transporters and their regulatory genes LXRalpha, PPARgamma, and SREBPs were found. ABCA1, ABCG1, and ABCA7 proteins were localized in glandular MG epithelial cells (MEC) during lactation, whereas during nonlactating stages, depending on species, the proteins showed distinct localization patterns in MEC and adipocytes. Our results demonstrate that ABCA1, ABCG1, and ABCA7 are differentially expressed between lactation and nonlactating stages and in association with regulatory genes. Combined expression and localization data suggest that the selected cholesterol transporters are universal MG transporters involved in transport and storage of cholesterol and in lipid homeostasis of MEC. Because of the species-specific expression patterns of transporters in mammary tissue, mechanisms of cholesterol homeostasis seem to be differentially regulated between species.

Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

Immunohistochemical localization of osteoclastogenic cell mediators in feline tooth resorption and healthy teeth

Tooth resorption is among the most common and most challenging problems in feline dentistry It is a progressive disease eventually leading to tooth loss and often root replacement. The etiology of moth resorption remains obscure and to date no effective therapeutic approach is known. The present study is aimed at assessing the reliability of radiographic imaging and addressing the possible involvement of receptor activator of NF kappa B (RANK), its ligand (RANKL), and osteoprotegerin (OPG) in the process of tooth resorption. Teeth from 8 cats were investigated by means of radiographs and paraffin sections followed by immunolabeling. Six cats were diagnosed with tooth resorption based on histopathologic and radiographic findings. Samples were classified according to a four-stage diagnostic system. Radiologic assessment of tooth resorption correlated very strongly with histopathologic findings. Tooth resorption was accompanied by a strong staining with all three antibodies used, especially with anti-RANK and anti-RANKL antibodies. The presence of OPG and RANKL at the resorption site is indicative of repair attempts by fibroblasts and stromal cells. These findings should be extended by further investigations in order to elucidate the pathophysiologic processes underlying tooth resorption that might lead to prophylactic and/or therapeutic measures. J Vet Dent 27(2); 75 - 83, 2010

The microtubule plus-end localization of Aspergillus dynein is important for dynein-early-endosome interaction but not for dynein ATPase activation

Cytoplasmic dynein in filamentous fungi accumulates at microtubule plus-ends near the hyphal tip, which is important for minus-end-directed transport of early endosomes. It was hypothesized that dynein is switched on at the plus-end by cargo association. Here, we show in Aspergillus nidulans that kinesin-1-dependent plus-end localization is not a prerequisite for dynein ATPase activation. First, the Walker A and Walker B mutations in the dynein heavy chain AAA1 domain implicated in blocking different steps of the ATPase cycle cause different effects on dynein localization to microtubules, arguing against the suggestion that ATPase is inactive before arriving at the plus-end. Second, dynein from kinA (kinesin 1) mutant cells has normal ATPase activity despite the absence of dynein plus-end accumulation. In kinA hyphae, dynein localizes along microtubules and does not colocalize with abnormally accumulated early endosomes at the hyphal tip. This is in contrast to the colocalization of dynein and early endosomes in the absence of NUDF/LIS1. However, the Walker B mutation allows dynein to colocalize with the hyphal-tip-accumulated early endosomes in the kinA background. We suggest that the normal ability of dyenin to interact with microtubules as an active minus-end-directed motor demands kinesin-1-mediated plus-end accumulation for effective interactions with early endosomes.

Cardiogoniometric parameters for detection of coronary artery disease at rest as a function of stenosis localization and distribution

Cardiogoniometry (CGM), a spatiotemporal electrocardiologic 5-lead method with automated analysis, may be useful in primary healthcare for detecting coronary artery disease (CAD) at rest. Our aim was to systematically develop a stenosis-specific parameter set for global CAD detection. In 793 consecutively admitted patients with presumed non-acute CAD, CGM data were collected prior to elective coronary angiography and analyzed retrospectively. 658 patients fulfilled the inclusion criteria, 405 had CAD verified by coronary angiography; the 253 patients with normal coronary angiograms served as the non-CAD controls. Study patients--matched for age, BMI, and gender--were angiographically assigned to 8 stenosis-specific CAD categories or to the controls. One CGM parameter possessing significance (P < .05) and the best diagnostic accuracy was matched to one CAD category. The area under the ROC curve was .80 (global CAD versus controls). A set containing 8 stenosis-specific CGM parameters described variability of R vectors and R-T angles, spatial position and potential distribution of R/T vectors, and ST/T segment alterations. Our parameter set systematically combines CAD categories into an algorithm that detects CAD globally. Prospective validation in clinical studies is ongoing.

Localization of primary language areas by arcuate fascicle fiber tracking

To reduce the risk of disabling postoperative functional deficit in patients with lesions in the dominant hemisphere, information about the localization of eloquent language areas is mandatory.

Expression and localization pattern of ABCA1 in diverse human placental primary cells and tissues

The ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes. Recent data provide evidence that ABCA1 plays an important role in placental function but the exact cellular sites of ABCA1 action in the placenta remain controversial. To clarify this issue, we analyzed the cellular and subcellular localization of ABCA1 with immunocytochemistry, immunofluorescence and subsequent confocal or immunofluorescence microscopy in different types of isolated primary placenta cells: cytotrophoblast cells, amnion epithelial cells, villous macrophages (Hofbauer cells), and mesenchymal cells isolated from chorionic membrane and placental villi. After 12 h of cultivation, primary cytotrophoblast cells showed intensive membrane and cytoplasmic staining for ABCA1. After 24 h, with progressive syncytium formation, ABCA1 staining intensity was markedly reduced and ABCA1 was dispersed in the cytoplasm of the forming syncytial layer. In amnion epithelial cells, placental macrophages and mesenchymal cells, ABCA1 was predominantly localized at the cell membrane and cytoplasmic compartments partially corresponding to the endoplasmic reticulum. In these cell types, the ABCA1 staining intensity was not dependent on the cultivation time. In conclusion, ABCA1 shows marked expression levels in diverse placental cell types. The multitopic localization of ABCA1 in diverse human placental cells not all directly involved in materno-fetal exchange suggests that this protein may not only participate in transplacental lipid transport but could have additional regulatory functions.

A shortened radiofrequency denervation method for cervical zygapophysial joint pain based on ultrasound localization of the nerves

Radiofrequency neurotomy is a recognized treatment for cervical zygapophysial joint pain. In several studies, the method has provided complete pain relief in 60-70% of the patients for approximately 9 months. The validated technique has the disadvantage of procedural times of 2-4 hours because several lesions are performed to take into account the variable nerve course. We tested the hypothesis that ultrasound localization of the nerves would enable us to reduce the number of lesions performed, while reaching the benchmark of at least 80% pain relief in 80% of patients with a median duration of 35 weeks, as achieved by a previous investigation using the standard method.

Typical 3-D localization of tumor remnants of WHO grade II hemispheric gliomas--lessons learned from the use of intraoperative high-field MRI control

Complete resection of grade II gliomas might prolong survival but is not always possible. The goal of the study was to evaluate the location of unexpected grade II gliomas remnants after assumed complete removal with intraoperative (iop) MRI and to assess the reason for their non-detection.