Marker Dye Research Articles

A cytosolic phospholipase A2-like protein in the filamentous fungusAspergillus oryzaelocalizes to the intramembrane space of the mitochondria

In mammalian cells, cytosolic phospholipase A(2) (cPLA(2)) displays a variety of activities through the release of arachidonic acid in response to cellular stimuli. In this study, we characterize the putative cPLA(2)-like protein, AoPlaA, in the filamentous fungus Aspergillus oryzae. When AoPlaA-EGFP was expressed in A. oryzae, it localized to the tubular structures that was costained by the marker dye for the mitochondria. A biochemical fractionation experiment showed that AoPlaA was present in the mitochondria-enriched fraction. The presence of an N-terminal cleavable mitochondrial targeting signal in AoPlaA was demonstrated by N-terminal amino acid sequence analysis, and by showing that chimeric proteins consisting of N-terminal 65 or 50 amino acids of AoPlaA fused to enhanced green fluorescent protein (EGFP) localized to the mitochondria. Submitochondrial fractionation of AoPlaA expressed in Saccharomyces cerevisiae demonstrated that AoPlaA localizes to the intramembrane space of the mitochondria. Taken together, the results presented here demonstrate a novel, submitochondrial localization of the cPLA(2)-like protein in the filamentous fungi.

Sentinel lymph node mapping as a side-effect of colonoscopic tattooing

Correct tumor localization is crucial for proper surgical therapy in colorectal cancer. Intraoperative visualization of the lesion is facilitated by preoperative colonoscopic tattooing, regardless of whether an open or laparoscopic approach is employed. This pilot study tests the hypothesis that colonoscopic tattooing can serve the additional role of sentinel lymph node (SLN) mapping. We collected 5 prospective and 16 retrospective cases, in which colonoscopic tattooing was applied and surgery was performed. Nineteen of these cases showed colorectal cancer. High-grade intraepithelial neoplasia was found in two cases. All lymph nodes (LNs) were histologically assessed for metastasis and carbon particles, and those that tested positive were registered as carbon-containing lymph nodes (CcLNs). Subsequently, additional step sections were cut and immunohistochemistry was performed on all lymph nodes of the malignant cases. A total number of 311 lymph nodes were investigated. CcLNs could be identified in 17 of 21 cases (detection rate: 81%). The histomorphology of CcLNs was identical to that known from carbon as a sentinel marker dye. The mean CcLN number was 2 +/- 2 (range 1-6). After primary evaluation, one metastasis was detected in a case where a CcLN was not observed. All other cases showed no positive LNs. After step sectioning and immunohistochemical staining, one additional micrometastasis was found in a CcLN, resulting in upstaging from N0 to N1 (mi). Our findings support the thesis that colonoscopic tattooing holds the potential for SLN mapping. Therefore, a prospective study with an appropriate case number should follow this pilot study to clarify the clinical value of this finding.

Confocal Microscopy Reveals Zones of Membrane Remodeling in the Outer Cortex of the Human Lens

To optimize fixation, sectioning, and immunolabeling protocols to map the morphology of the human lens with confocal microscopy. Transparent human lenses were fixed in 0.75% paraformaldehyde for 24 hours, cut in half, and fixed for another 24 hours. Lenses were cryoprotected, sectioned, and labeled with wheat germ agglutinin, aquaporin-0 antibodies, Hoechst, or toluidine blue. Before fixation, some lenses were incubated in an extracellular marker dye, Texas Red-dextran. Labeled sections were imaged with a confocal microscope. Overlapping images were tiled together to form a continuous image montage of fiber cell morphology from the periphery to the lens center. Fiber cell morphologies were identical with those previously described by electron microscopy and allowed immunohistochemistry to be performed for a representative membrane protein, aquaporin-0. Sectioning protocols enabled the epithelium and outer cortex to be retained, leading to the identification of two unique morphologic zones. In the first zone, an age-independent compaction of nucleated fiber cells and the initiation of extensive membrane remodeling occur. In the second zone, fiber cells retain their interdigitations but lose their nuclei, exhibit a distorted shape, and are less compressed. These zones are followed by the adult nucleus, which is marked by extensive compaction and a restriction of the extracellular space to the diffusion of Texas Red-dextran. The authors have developed sectioning and imaging protocols to capture differentiation-dependent changes in fiber cell morphology and protein expression throughout the human lens. Results reveal that differentiating fiber cells undergo extensive membrane remodeling before their internalization into the adult nucleus.

Possible involvement of PPARγ in the regulation of basal channel opening of P2X7 receptor in cultured mouse astrocytes

Aims Recently, we demonstrated that cultured mouse astrocytes exhibited basal channel opening of P2X7 receptor (P2X7R) in the absence of any exogenous ligand, but the regulatory mechanism involved was not elucidated. Since our preliminary experiments suggested possible involvement of peroxisome proliferator-activated receptor (PPAR) γ in the regulation, we examined whether PPARγ regulated P2X7R basal channel opening in mouse astrocytes. Main methods P2X7R channel opening was assessed as to the uptake of a marker dye, YO-PRO-1 ® (YP), in the presence or absence of agonists and antagonists for PPARγ under a fluorescence microscope. Expression of PPARγ was evaluated by Western blotting and immunocytochemistry. Key findings NSAIDs such as flufenamic acid (FFA) and indomethacin, which are a cyclooxygenase inhibitor and a PPARγ agonist, showed enhancing and inhibiting effects on YP uptake at low and high concentrations, respectively, and the enhanced uptake was abolished by periodate-oxidized ATP (oxATP), a selective P2X7R antagonist. The PPARγ agonists 15-deoxy- Δ 12,14-prostaglandin J 2 and ciglitazone decreased the basal and FFA-enhanced YP uptake, while the antagonist GW9662 increased YP uptake, this effect being blocked by the agonists and also by oxATP. PPARγ was distributed in the nucleus and cytosolic/membrane fraction of cultured mouse astrocytes. Significance These findings indicate that basal channel opening of P2X7R in mouse astrocytes is at least in part regulated by PPARγ.

Using Image Analysis to Design a Prototype Autodissemination Device Intended for the Biorational Control of <I>Plodia Interpunctella</I>

The extent to which a carrier powder was taken up and horizontally transferred by contaminated Indianmeal moth, Plodia interpunctella (Hübner) individuals to conspecifics was evaluated. Using a marker dye, which was shown to be colocalized with the carrier powder, the amount of powder on the Indianmeal moth, as determined by spectrophotometry, was correlated with quantification based on a novel image analysis approach that enabled differentiation of powder uptake by body region. Over a 48-h period, more powder was retained on the ventral surface than the dorsal side, with the head region showing the greatest amount of powder uptake and retention. During courtship, powder was horizontally transferred by treated males to 1.6% of the head and 0.06% of the untreated females' body. To inoculate moths, a pit-fall style autodissemination station was determined as being more effective than a powder tray, as significantly more material was taken up to the key areas of the moth's body where powder is more effectively retained and more likely to be horizontally transferred. Additionally, a pit-fall station prevented moths from exhibiting avoidance behavior from the powder, which was frequently encountered if they had to walk into a powder tray. This study shows that different regions of the Indianmeal moth body vary in capacity to carry powder and that future research efforts should target these specific regions. Although the proposed autodissemination system was optimized for management strategies of Indianmeal moth, we believe the results presented here can be used to develop novel management strategies for other insect pests.

Evaluation of Intraosseous Delivery of Factor VIIa during Hemorraghic Shock in the Pig

The purpose of this investigation is to determine if intraosseous infusion (IO) is a suitable method for the delivery of recombinant human factor VIIa (rFVIIa) during hemorrhagic shock. The measures that were used to evaluate IO delivery suitability included: (1) determination of clinically significant local or systemic toxicity and (2) demonstration that systemic blood levels of rFVIIa increased rapidly following administration. Our results indicate that there was no evidence of significant local or systemic toxicity following infusion and that the systemic blood concentration of rFVIIa peaks immediately after the end of infusion. This result suggests that the systemic blood level profiles of rFVIIa delivered by IO infusion are similar to those that could be produced by intravenous (IV) administration. Furthermore, in all 25 test animals, access to the systemic circulation during shock was achieved as evidenced by rapid increase in a marker dye (flourescein) or rFVIIa in the blood. We conclude that administration of rFVIIa via IO infusion is a reasonable safe method and is likely to produce blood levels required for improved hemostasis during shock.

Reducing Laparoscopic Radical Prostatectomy False-Positive Margin Rates Using Cyanoacrylate Tissue Glue

Trauma to the prostate surface from laparoscopic instruments may have a role in creating false-positive margins during laparoscopic radical prostatectomy (LRP). To determine the feasibility of using cyanoacrylate glue to repair iatrogenic lacerations and to evaluate the glue's effect on the positive surgical margin rates of LRP specimens. We used porcine kidneys as a surrogate experimental tissue to help determine the quality, robustness, and adequacy of glued repairs in experimentally created lacerations. A matched control group of unrepaired kidney specimens and kidney specimens repaired with glue were subjected to full histopathologic processing. Exposure of a nephron to surface marker ink was considered to be a "positive margin." The efficacy and impact of glue repairs on LRP specimens that had sustained iatrogenic intraoperative surface trauma were also assessed. We evaluated the success of glue repair in preventing subcapsular renal parenchymal staining. We also compared the rate of positive margins in LRP specimens with and without routine glue repair of the surface of the prostate. The glue remained effective throughout the entire laboratory process and did not interfere with histopathologic assessment. As hypothesised, cyanoacrylate glue repair of the renal lacerations prevented staining of subcapsular tissues with marker dye and therefore prevented what might otherwise be considered false-positive staining. The rate of positive margins of the 40 LRP specimens without glue repair was 35%, compared with a rate of 10% for 40 glue-repaired specimens. The limitations of the study are that follow-up was short and that the prostatectomy specimens were compared with consecutive controls rather than with matched randomised controls. Cyanoacrylate glue is a novel, inexpensive, and very effective prostate repair agent that does not interfere with histologic processing. It is possible to accurately repair iatrogenic prostate lacerations with cyanoacrylate glue and, in doing so, to considerably reduce the rate of spurious false-positive surgical margins.

An Experimental Approach to Measure Mass Diffusion in Rat Tumor Tissue

The objective of this research is to evaluate the usefulness of a macroscopic, fluorescent, imaging technique to quantify spatiotemporal mass transport parameters in in vitro solid tumor tissues taken from rat models. Fluorescent images captured during the experiments are digitally analyzed to determine the concentration of a fluorescent marker dye as it diffuses into tissue specimens taken from rat tumors. The collected concentration data are used to estimate local diffusion coefficients. An analysis of the distribution of the local diffusion data indicates that the local diffusion coefficient is spatially dependent within the tumor tissue. When mass transfer is restricted to one dimension, the current technique can be used to determine the concentration distribution of fluorescent molecules on the tissue surface and to estimate the mass transfer parameters within the heterogeneous tumor tissue.

Effect of Superoxide Derived from Lucifer Yellow CH on Voltage-Gated Currents of Mouse Taste Bud Cells

Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.

Aldosterone acutely increases the production of reactive oxygen species (ROS) and intracellular calcium concentration [Ca2+]i in neurons of the paraventricular nucleus (PVN): An in vitro study

Besides the well known genomic or transcriptional effects of aldosterone (ALDO), non‐transcription‐like (rapidly acting) actions have been recently demonstrated. We investigated the effects of ALDO on changes in [Ca2+]i and ROS production in PVN cells in vitro. Neurons in the PVN were visualized by confocal microscopy. Changes in [Ca2+]i were measured as changes in the fluorescence of Fluo‐4 AM, a [Ca2+]i marker dye. To estimate ROS, we measured the changes in the ethidium fluorescence which is directly proportional to the oxidation of dihydroethidium (DHE) by ROS. Male rats were decapitated and the brains were promptly removed, cooled in an artificial cerebrospinal fluid (aCSF) containing 7.5% sucrose and cut on a vibratome. The cells were loaded with Fluo‐4 (18 μM) or DHE (20 μM) added to oxygenated aCSF (34±0.5°C) for 1h. The slices were then placed in a chamber for microscopic visualization of fluorescence. The chamber was perfused with heated and oxygenated aCSF and ALDO (1 μM) or KCl (60 mM). Three‐dimensional images were obtained every minute for 1h. The perfusion of ALDO increased both [Ca2+]i (Δ=69.9±10.6%) and ROS (Δ=26.0±2.5%) within 15–30 min in the PVN neurons (i.e., KCl responsive cells). The increase in [Ca2+]i suggests that ALDO can acutely activate neurons in the PVN, and the enhanced ROS production implies that ROS may mediate a non‐transcriptional effect of ALDO. (Support: NIH HL‐59676 & HL‐62261)

Phenotypic changes of morphologically identified guinea‐pig myenteric neurons following intestinal inflammation

We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca(2+)-dependent K(+) channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (I(h)) antagonist Cs(+). It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

Variable growth band deposition leads to age and growth uncertainty in the western wobbegong shark, Orectolobus hutchinsi

Age and growth parameters of Orectolobus hutchinsi were estimated using micro-radiographs of sectioned vertebrae from 182 wild caught individuals. Two fluorochrome marker dyes, calcein and oxytetracycline, were used to validate the timing and periodicity of vertebral band formation in nine individuals held in the laboratory for between 423 and 472 days. Growth bands were difficult to interpret and final counts were obtained from only 98 (53.8%) individuals ranging in total length (TL) from 63 to 146 cm. The timing of growth band formation in the vertebrae of captive animals had no predictable temporal pattern, with formation occurring during all seasons of the year, making age validation difficult. Growth band formation was hypothesised to be influenced by non-periodic changes in centrum or somatic growth rather than seasonal growth, as observed in many other elasmobranch species. Growth rates of nine O. hutchinsi held in captivity varied considerably, ranging from 3.5 cm year–1 to 13.8 cm year–1 in total length (mean = 7.03 cm year–1). Although the periodicity of vertebral band formation in captive animals did not support a synchronous annual pattern, captive growth rates matched those predicted when an annual band pattern was assumed for wild caught individuals. Von Bertalanffy growth parameters estimated from vertebral analysis assuming an annual banding pattern and a mean size of birth of 24.1 cm were: L∞ = 149.45 cm and K = 0.117 year–1 for both sexes combined. These results illustrate the fundamental importance of validating the periodicity of growth band formation in elasmobranch age and growth studies as it has considerable implications for the management of fisheries that exploit shark and ray species that may exhibit asynchronous growth band deposition.

Aluminium toxicity in plants: internalization of aluminium into cells of the transition zone in Arabidopsis root apices related to changes in plasma membrane potential, endosomal behaviour, and nitric oxide production

The extent of aluminium internalization during the recovery from aluminium stress in living roots of Arabidopsis thaliana was studied by non-invasive in vivo microscopy in real time. Aluminium exposure caused rapid depolarization of the plasma membrane. The extent of depolarization depends on the developmental state of the root cells; it was much more extensive in cells of the distal than in the proximal portion of the transition zone. Also full recovery of the membrane potential after removal of external aluminium was slower in cells of the distal transition zone than of its proximal part. Using morin, a vital marker dye for aluminium, and FM4-64, a marker for endosomal/vacuolar membranes, an extensive aluminium internalization was recorded during the recovery phase into endosomal/vacuolar compartments in the most aluminium-sensitive cells. Interestingly, aluminium interfered with FM4-64 internalization and inhibited the formation of brefeldin A-induced compartments in these cells. By contrast, there was no detectable uptake of aluminium into cells of the proximal part of the transition zone and the whole elongation region. Moreover, cells of the distal portion of the transition zone emitted large amounts of nitric oxide (NO) and this was blocked by aluminium treatment. These data suggest that aluminium internalization is related to the most sensitive status of the distal portion of the transition zone towards aluminium. Aluminium in these root cells has impact on endosomes and NO production.

Ca2+ sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers

Ca(+) sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca(2+) sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca(2+) sparks were rare in these fibers. At 5-7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca(2+) sparks. The average rate of occurrence of Ca(2+) sparks is >10-fold higher after 5-7 days in culture than in days 1-3. With the use of fibers cultured for 7 days, application of the Ca(2+) channel blockers Co(2+) or nifedipine almost completely suppressed the occurrence of Ca(2+) sparks, as previously shown in embryonic fibers, suggesting that Ca(2+) sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca(2+) sparks, suggesting that Ca(2+) sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling.

Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-α-d-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II

Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-alpha-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring d-xylose from UDP-alpha-d-xylose to l-fucose. The disaccharide product was hydrolyzed by alpha-xylosidase, whereas no reaction was catalyzed by beta-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an alpha-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-alpha-d-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A-sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-d-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.

Photoinduced Phase Separation

A novel approach of photoinduced phase separation has been demonstrated with a photolabile anionic surfactant, mixed with an inert nonionic surfactant in the presence of salting-out electrolyte. Breakdown of the photolyzable surfactant results in hydrophobic photoproducts, which are emulsified by the remaining inert surfactant; added electrolyte resolves the emulsion into macroscopic oily and aqueous phases. The initial micellar systems can disperse an insoluble additive marker dye (shown), which may be spatially segregated from the aqueous environment by the action of UV light.

Endocytosis and vesicle trafficking during tip growth of root hairs

The directional elongation of root hairs, "tip growth", depends on the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material. So far, little is known about the dynamics of endocytosis in living root hairs. We analyzed the motile behaviour of vesicles in the apical region of living root hairs of Arabidopsis thaliana and of Triticum aestivum by live cell microscopy. For direct observation of endocytosis and of the fate of endocytic vesicles, we used the fluorescent endocytosis marker dyes FM 1-43 and FM 4-64. Rapid endocytosis was detected mainly in the tip, where it caused a bright fluorescence of the apical cytoplasm. The internalized membranes proceeded through highly dynamic putative early endosomes in the clear zone to larger endosomal compartments in the subapical region that are excluded from the clear zone. The internalized cargo ended up in the dynamic vacuole by fusion of large endosomal compartments with the tonoplast. Before export to these lytic compartments, putative early endosomes remained in the apical zone, where they most probably recycled to the plasma membrane and back into the cytoplasm for more than 30 min. Endoplasmic reticulum was not involved in trafficking pathways of endosomes. Actin cytoskeleton was needed for the endocytosis itself, as well as for further membrane trafficking. The actin-depolymerizing drug latrunculin B modified the dynamic properties of vesicles and endosomes; they became immobilized and aggregated in the tip. Treatment with brefeldin A inhibited membrane trafficking and caused the disappearance of FM-containing vesicles and putative early endosomes from the clear zone; labelled structures accumulated in motile brefeldin A-induced compartments. These large endocytic compartments redispersed upon removal of the drug. Our results hence prove that endocytosis occurs in growing root hairs. We show the localization of endocytosis in the tip and indicate specific endomembrane compartments and their recycling.

Extended Therapeutic Window and Functional Recovery after Intraarterial Administration of Neuregulin-1 after Focal Ischemic Stroke

We have previously shown that neuregulin-1 (NRG-1) protects neurons from ischemic brain injury if administered before focal stroke. Here, we examined the therapeutic window and functional recovery after NRG-1 treatment in rats subjected to 90 mins of middle cerebral artery occlusion (MCAO) and 24 h of reperfusion. Neuregulin-1 (2.5 microg/kg [corrected] bolus, 1.25 microg/kg/min [corrected] infusion) reduced infarct volume by 89.2%+/-41.9% (mean+/-s.d.; n=8; P<0.01) if administered immediately after the onset of reperfusion. Neuroprotection was also evident if NRG-1 was administered 4 h (66.4%+/-52.6%; n=7; P<0.01) and 12 h (57.0%+/-20.8%; n=8; P<0.01) after reperfusion. Neuregulin-1 administration also resulted in a significant improvement of functional neurologic outcome compared with vehicle-treated animals (32.1%+/-5.7%; n=9; P<0.01). The neuroprotective effect of the single administration of NRG-1 was seen as long as 2 weeks after treatment. Neurons labeled with the neurodegeneration marker dye Fluoro-JadeB were observed after MCAO in the cortex, but the numbers were significantly reduced after NRG-1 treatment. These results indicate that NRG-1 is a potent neuroprotective compound with an extended therapeutic window that has practical therapeutic potential in treating individuals after ischemic brain injury.

1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells

Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (32P). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)2D3 resulted in an apparent increase in 32P uptake within 1 min, relative to controls, that was statistically significant from 3-10 min of incubation. Using the endocytic marker dye, FM1-43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical> basal) within 10 s of hormone treatment, a return of fluorescence at 15-65 s, followed by another round of decreasing and increasing fluorescence. Between 3-9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)2D3 (1,25D3-MARRS) inhibited both 32P uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both 32P uptake and the observed 1,25(OH)2D3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)2D3-stimulated vesicular trafficking is mediated by the 1,25D3-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.

Structural Properties of Oligonucleotide Monolayers on Gold Surfaces Probed by Fluorescence Investigations

We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.