N A N O P R O B E S E - N E W S
Vol. 9, No. 10 October 31, 2008
Updated: October 31, 2008
In this Issue:
This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful, as always, let us know if we can improve anything.
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As we have reported previously, the small size of our Nanogold® labels makes them ideal for imaging the 3-dimensional distribution of proteins or other targets in cells or tissues by electron tomography. Nanogold-labeled conjugates typically display:
- High penetration into cells and tissues.
- High labeling density.
- Close to quantitative labeling of antigenic sites.
- High labeling resolution.
The only problem is that their small size can make them difficult to detect by conventional bright-field electron tomography, particularly in thicker sections. However, in a new paper in Nature, He, Björkman and co-workers use gold enhancement to enlarge the Nanogold for better visibility in their studies of FcRn-mediated antibody transport.
Upper: Resolution advantage: size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. Due to its position at the hinge region, Nanogold is positioned closer to the target upon binding, yet does not hinder or interfere with binding. Lower: Mechanism of enhancement of Nanogold® by GoldEnhance. Final particle size is controlled by enhancement time.
The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, and provides the fetus or newborn with humoral immunity before its own immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. This is facilitated by the pH difference between the apical (pH 6.06.5) and basolateral (pH 7.4) sides of intestinal epithelial cells: FcRn binds IgG at pH 6.06.5 but not at pH 7 or more, hence transport is unidirectional. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). The authors used electron tomography to visualize jejunal transcytosis directly in space and time, and to do so, developed new labeling and detection methods to map individual Nanogold-labeled Fc within transport vesicles, while simultaneously characterizing these vesicles by immunolabeling.
To prevent ligand misdirection caused by a bulky label, the authors covalently attached small (1.4-nm) Monomaleimido Nanogold to IgG-Fc (Au-Fc) at a site distant from where FcRn binds. Optimum conditions to selectively reduce hinge disulfides were obtained by evaluating the results of different reduction protocols using Ellmans Reagent. 1.01.5 mg of Fc was reduced with 1218 mg of mercaptoethylamine hydrochloride (MEA) in 1 mL of 0.1 M NaPO4, pH 6.0, 5 mM EDTA for 1.5 hours at 37°,C, then passed over a Superdex-75 size exclusion column in 20 mM NaPO4, pH 6.5 with 150 mM NaCl and 1 mM EDTA. After concentration, the reduced Fc was labeled with Monomaleimido Nanogold, which reacts specifically with reduced sulfhydryls: ~30 nmol of Monomaleimido Nanogold was suspended in 1 mL deionized water, then immediately incubated with the reduced Fc for 24 hours at room temperature. Labeled Fc (AuFc) was separated from unlabeled Fc and free Nanogold by gel filtration using the Superdex 75 column. Labeled Fc was further purified by passage over an FcRn affinity column at pH 6, followed by elution at pH 8. Molar concentrations of Nanogold and Fc were determined spectrophotometrically using absorptions at 420 and 280 nm respectively, using extinction coefficients of 155,000 M-1cm1 for Nanogold and 60,900 M-1cm1 for Fc. The monofunctional nature of the Nanogold reagent avoids multiple labeling, which could erroneously prolong release through avidity. For steady-state experiments, Au-Fc was fed to neonatal rats, rather than incubated with excised intestines, which causes morphological changes: 1113-day-old suckling rats were fed with three 100-µL doses of Au-Fc or Au-dextran (about 3 mM), after which the small intestine was harvested for chemical or cryofixation. For kinetic experiments, ligated intestines were incubated with 3 µMAu-Fc (pulse experiments), followed by unlabelled Fc or IgG (pulsechase experiments); immunolabeling was performed on gold-enhanced intestinal samples with a modification of the Tokuyasu method for immunolabeling cryosections.
Intestinal segments were chemically fixed and treated with GoldEnhanceEM, then fixed and stained with OsO4 and uranyl acetate described previously. Traditional pre-embedding enhancement protocols are incompatible with freeze-substitution fixation, therefore, the authors used previously reported methods for enlarging small gold clusters in the cold organic solvents. After HPF of intestinal segments, a three-step enlarging protocol involving silver enhancement was used to slightly enlarge the Nanogold (to at most 8 nm), followed by coating the silver shell with gold to make it impervious to osmium, then enlargement to 10 16 nm by using gold enhancement. Samples were then treated with OsO4 and uranyl acetate, and warmed to room temperature (about 22°C). HPF/FSF or chemically fixed samples were infiltrated with resin, polymerized, cut into 120200-nm sections and examined with an FEI T12 transmission electron microscope operating at 120 kV. For tomography, dual-axis tilt series were collected at X 6,500 from 120 - 200 nm sections at 700nm underfocus. Tomograms were computed for each tilt axis by using enlarged golds as alignment markers, and then aligned and combined to form a dual-axis tomogram using IMOD. Tomographic slices were usually 1.6nm thick. Tomographic reconstructions were interpreted and modeled by manual segmentation with IMOD.
Combining electron tomography with a nonperturbing endocytic label enabled the conclusive identification of receptor-bound ligands. Interconnecting vesicles were successfully resolved, it could be determined whether individual vesicles were microtubule-associated, and FcRn-mediated transport of IgG could be accurately traced. The results of this study present a complex system, in which Fc moves through networks of entangled tubular and irregular vesicles, of which only some are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis were elucidated, including transport involving multivesicular body inner vesicles/tubules, and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes were each used to vesicles in different and overlapping morphological classes, revealing the spatial complexity of endo-lysosomal
- He, W.; Ladinsky, M. S.; Huey-Tubman, K. E.; Jensen, G. J.; McIntosh, J. R., and Björkman P. J.: FcRn-mediated antibody transport across epithelial cells revealed by electron tomography. Nature, 455, 542-546 (2008).
In some situations, GoldEnhance may produce a fine, "granular" non-specific background signal of small particles. There are several ways in which you can either slow down development so that you have more control over its progress, or chemically "stop" the development process. Which approach is best for the system under study depends upon whether you believe the primary problem is continued development after rinsing, in which case a chemical "stop" is appropriate, or excessively fast development before rinsing, in which case slowing down the reaction is most appropriate. We have extensively discussed alternative strategies for controlling background in a previous article and in our technical support page for GoldEnhance.
If you are observing fine background that seems to arise from continued development after rinsing to remove the GoldEnhance reagents, a number of "stop" procedures are available. The simplest and most universal is treatment with 1% or 2% freshly prepared sodium thiosulfate for a few seconds, and unless there are specific reasons that this would be harmful to your specimen, we usually suggest that you try this first.
Wanzhong He, from the Bjorkman Laboratory in the Division of Biology at the California Institute of Technology has been kind enough to send us some results using a combination of methods that eliminated background and yielded very clean staining:
- A ratio of one part solution A (enhancer) : 5 parts solution B (activator) was used.
- Solution D (buffer) was substituted with 0.05 M sodium phosphate with 0.1 M sodium chloride, pH 5.5.
- Enhancement was performed for 10-15 minutes rather than 3 minutes.
- The reaction was "stopped" using 3% Na2S2O3 for 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes.
Some images that demonstrate the effect of this procedure are shown below:
left: Gold enhancement of intestinal tissue with Nanogold staining. (a) Nanogold particles, then enhanced with GoldEnhance EM for 3 minutes, (b) control with Nanogold labeling omitted, showing the granular background. right: analogous procedure, using modified protocol to control background, comprising: one part A (enhancer) : 5 parts B (activator), D (buffer) was substituted with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5, enhancement was performed for 10-15 minutes rather than 3 minutes, "stopped" using 3% Na2S2O3 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes. (c) Nanogold particles, enhanced with GoldEnhance EM, (d) control with Nanogold labeling omitted, identical gold enhancement procedure. Insets show experimental (left) and control (right) specimens (Thanks to Wanzhong He and Dr. Pamela Bjorkman for the images).
- He, W.; Kivork, C.; Machinani, S.; Morphew, M. K.; Gail, A. M.; Tesar, D. B.; Tiangco, N. E.; McIntosh, J. R.; and Bjorkman, P. J.: A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules. J. Struct. Biol., 160, 103-113 (2007).
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When labeling with Monomaleimido Nanogold®, it is often necessary to reduce a disulfide group to generate a thiol for labeling. This can be one of the most tricky parts of the procedure, and can present a number of challenges:
Generation of Fab' fragments from whole IgG molecule, and site-specific labeling of a hinge thiol in a Fab fragment with a Monomaleimido gold cluster.
- Sometimes multiple disulfides are present (for example, hinge and intra-chain disulfides in antibodies) and it is necessary to selectively reduce one while leaving the others intact.
- Disulfides can vary in their reductive behavior even in quite similar molecules, such as IgG from different species, or monoclonal vs. polyclonal antibodies.
- Atmospheric oxygen can re-oxidize reduced thiols, especially if traces of transition metal ions such as iron are present that can catalyze the reaction. To prevent this, observe the following precautions:
- Use plastic rather than metal spatulas to handle, weigh and dispense chemicals.
- Thoroughly degass all buffers and solvents immediately before use.
- Include 5 mM disodium dihydrogen ethylene diamine tetraacetic acid (EDTA) in the reaction buffer, and 1 mM in the buffer used to separate the reduced antibody or biomolecule. This will chelate any transition metal ions and help sequester them from reaction with atmospheric oxygen and reduced disulfides.
- Conduct reductions in a sealed vial under an inert atmosphere such as nitrogen or argon.
- The accessibility of the thiol that is generated should be considered, if possible by examining the known structure of the biomolecule to be labeled.
- The effect of disulfide reduction on the integrity and activity of the molecule may require consideration, especially in small molecules such as peptides, or if multiple disulfides are to be reduced.
With polyclonal antibodies, our general experience has been that those raised in goat are somewhat more easily reduced than those raised in mouse or expecially rabbit. We recommend using either dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA) at pH 6.0 (in 0.1 M sodium phosphate buffer) for antibody reduction: typically, we have found that goat antibodies are reduced by 20 mM reducig agent, while mouse or rabbit antibodies require 50 mM. However, these values are subject to change, and should be viewed as starting points rather than specific directions. We recommend reducing only a small portion of your antibody or biomolecule first, and determining whether the reaction was successful before reducing the balance.
A number of methods exist to assay thiols, and you can use these to determine whether your reduction was successful:
- A sensitive colorimetric procedure using dithiopyridines. Reference:
- Grassetti, D. R., and Murray, J. F. Jr.: Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine. Arch. Biochem. Biophys., 119, 41-49 (1967).
- 14C iodoacetic acid radiolabeling.
Separation of the reduced antibody or biomolecule from the excess reducing agent can also be an issue. Although with antibodies and proteins this is usually straightforward using gel filtration, it can be problematic for smaller molecules such as small peptides, or if you do not have access to chromatographic facilities. Complete separation is necessary because thiol-based reducing agents such as DTT or MEA will also react with maleimides to inactivate them and make them unreactive towards the reduced biomolecule. Even the relatively low concentrations typically used for antibody and protein reductions represent a large excess over the biomolecule, and even a very small amount of residual thiolated material can seriously compromise labeling. To ensure complete removal, we recommend gel filtration; other methods, particularly dialysis, do not give acceptable separation.
If you do not have access to gel filtration, then it may be possible to separate the reducing agent by repeated membrane centrifugation. However, this should be done thoroughly: we recommend at least five washes - and the material should be reduced in volume as completely as possible each time. In addition, it is essential to test the filtrate for the presence of thiols: the colorimetric procedure of Grassetti and Murray may be useful. Only when no thiol is detected in the filtrate is it safe to label the reduced biomolecule in the retentate with Monomaleimido Nanogold®.
Alternative reducing agents, particularly borohydrides such as sodium cyanoborohydride, have been used as reducing agents in place of thiols. While we have not investigated their use extensively, these offer an alternative to thiolated reagents, and because they do not react directly with maleimides, they may be used for reductions in which complete removal of the reducing agent from the reduced biomolecule is problematic.
Alternatively, you may consider labeling with Mono-Sulfo-NHS-Nanogold instead of Monomaleimido Nanogold. The labeling procedure with this reagent is simpler since no reduction is required, and almost all proteins and peptides have an accessible N-terminal amine for labeling. As described in a recent article, we find that even though labeling is not specific to the hinge region, antibodies labeled in this manner do not display any reduction in binding capacity.
Different reactions and Nanogold labeling reagents available for generating antibody fragments and Nanogold labeling.
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Conventional wisdom would dictate that atomic force microscopy (AFM) imaging requires a particle size of at least 2-4 nm. However, in their recent paper in Micron, Soman and group present a method using our 1.4 nm Nanogold® label for the Immunological identification of fibrinogen in dual-component protein films.
Implanted medical devices that are in contact with blood must have appropriate surface interactions with blood components. However, methods to study the interactions of such biomaterial surfaces with single molecules of blood components have been lacking, and techniques to measure single-molecule interactions in such complex multi-protein environments are therefore highly desirable. The authors demonstrate the use of atomic force microscopy in conjunction with Nanogold labels to detect the protein fibrinogen under aqueous conditions, without the topographical clues usually required for high-resolution visualization.
Mono-Sulfo-NHS-Nanogold was dissolved in 200 µL of deionized water. Polyclonal rabbit anti-human fibrinogen was then labeled with the dissolved Nanogold reagent using the procedure specified in the product instructions for labeling proteins of molecular weight greater than 15,000 Da. The anti-fibrinogen solution (1 mg/mL) was reacted with the activated Nanogold solution at pH 7.4 for 1 hour at room temperature while rotating on a hematology mixer to ensure optimal conjugation. The reaction mixture was then concentrated to a volume of 10 µL using a centricon centrifugal filter with a 30,000 molecular weight cut-off. The retentate was brought to a volume of 100 µL before purification by high performance liquid chromatography (HPLC) over a Superose 200 column (Beckman Coulter). The Nanogold conjugate was collected and stored at 4®C until required for use.
BSA was patterned onto both muscovite mica and plasma-treated polydimethylsiloxane (PDMS) substrates using micro-contact printing. A 15 mm diameter poly(dimethylsiloxane) rubber stamp having ~700 nm diameter holes was prepared by replication molding of a photoresist pattern prepared using optical lithography. The PDMS stamp was incubated in 1 or 5 mg/mL bovine serum albumin for 1 hour, rinsed in PBS for 5 minutes and dried with nitrogen. Freshly cleaved, hydrophilic, muscovite mica or glow-discharged plasma-cleaned PDMS was used as the substrate for stamping experiments. The PDMS substrate was rendered hydrophilic (water contact angle ~15°) by glow discharge plasma cleaning in an ambient environment for 45 minutes at 100 W power. A BSA-inked PDMS stamp was placed onto the mica or PDMS substrates under a 5 mg weight for 30 seconds to ensure proper stamping of proteins onto the substrate. The stamp was then carefully peeled off and the substrate surface immediately imaged using tapping mode AFM under PBS. The sample was transferred to an external fluid cell similar in design and dimension to the AFM fluid cell used for imaging. Human fibrinogen solution (1 mg/mL) was delivered through the fluid cell at a rate of 1 mL/h for 1 hour to allow fibrinogen to adsorb in the protein-free holes created by printing. Next, the sample was rinsed with buffer solution in the external fluid cell for 5 minutes: Nanogold-conjugated anti-fibrinogen (1 : 5 dilution) was then delivered to the fluid cell at a rate of 1 mL/h for 1 hour. The sample was again rinsed with PBS and moved to the AFM imaging stage for analysis.
All images were acquired in tapping mode (intermittent contact mode) with a Nanoscope IIIa Multimode1 AFM (Digital Instruments, CA) under both ambient and aqueous PBS. buffer using short thick cantilever silicon nitride probes (spring constant ~0.6 N/m2, Digital Instruments). Topographic images and phase images were captured at 512 x 512 pixels resolution with scan size 5 mm x 5 mm. Imaging was carried out at a scan rate of 1 Hz with a resonant frequency of ~8 kHz. The AFM probe free amplitude of oscillation and rsp values were fixed at 20 nm and 0.75 respectively for all images. AFM images were flattened using a first order line fit and low pass filtered to remove high frequency noise spikes from the images. Quantitative analysis of the dual-protein layer thickness was made using the crosssection tool provided with the instrument software.
protein patterns were visualized by tapping mode AFM after addition of conjugate. The Nanogold-conjugated antibody binds to the surface-adsorbed fibrinogen and is identified from the phase angle shift induced by the presence of the hard Nanogold particles. Control experiments using unlabeled antibody did not reveal a pattern by phase imaging, demonstrating that the conjugated gold particles were responsible for the image. Additional controls demonstrated that the pattern did not arise from migration of protein into the holes, or desorption of fibrinogen or BSA prior to or during labeling.
The new gold-antibody method offers numerous advantages over the previously reported adhesion force technique. Data can be acquired at much higher resolution (512 x 512 pixels vs. 32 x 32 pixels) and much faster acquisition times (8 minutes or less, vs. about 60 minutes for the adhesion technique) using standard AFM equipment. It is a direct imaging method and therefore does not require the complex statistical analyses necessary to interpret AFM force measurement images. This AFM immunodetection method has many potential applications to the evaluation of complex multi-component protein films adsorbed on clinically relevant polymers used in medical devices. It is particularly important because it can be used under conditions that model those found in vivo, and therefore affords a way to evaluate the performance of implantable medical devices under conditions close to those found in use. Potential applications include the study of competitive protein adsorption, conformation / functional changes in proteins on biomaterial surfaces, or analysis of the composition and makeup of the adsorbed proteins layer in retrieved biomedical implants.
- Soman, P.; Rice, Z., and Siedlecki, C. A. Immunological identification of fibrinogen in dual-component protein films by AFM imaging. Micron, 39, 832-842 (2008).
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Although not widely used, immunoenzymatic labeling is a viable electron microscopic labeling method, and enzymatically deposited DAB can provide sufficient contrast for electron microscopic imaging. DAN generally produces a relatively diffuse and continuous stain of medium to low contrast. Since this is easily differentiated from gold or silver-enhanced gold, the combination of enzymatic labeling with gold labeling provides a method for double labeling which has been used to generate significant results for a number of publications.
Left: schematic showing double labeling with DAB and silver-enhanced gold and the contrasting properties of the two labels. Right: Double labeling with silver-enhanced Nanogold and immunoperoxidase. Subcellular distribution of m4R immunoreactivity in striatal cholinergic neurons of control rats (A) or rats treated with oxotremorine (B). Cholinergic neurons were identified by immunoreactivity for ChAT, detected by the immunoperoxidase method: m4R reactivity was detected by Nanogold pre-embedding labeling with HQ silver enhancement. The ChAT immunoreaction product was visible throughout the cytoplasm of the perikarya. (A) The neuron immunopositive for ChAT and m4R has a large volume of cytoplasm, typical of a striatal interneuron, with immunoparticles at the external surface of the ER (arrows), Golgi apparatus (G), and external nuclear membrane ( flat arrow). Very few are associated with the plasma membrane (arrowheads). (B) After treatment with oxotremorine, most immunoparticles are clearly associated with endoplasmic reticulum lamina (arrows). Scale bars, 1 mm.
The latest contribution to the development of this method is described by Massi and colleagues in their recent Journal of Neuroscience paper on the role of cannabinoid receptors in the excitation of midbrain dopamine cells. The endocannabinoid system is involved in multiple physiological functions including reward. Cannabinoids are known to exert potent control over the activity of midbrain dopamine cells, but the contribution of cortical projections to this phenomenon is unclear. The authors hypothesized that cannabinoid type1 receptors (CB1-R) regulate the excitatory synaptic projections from the bed nucleus of the stria terminalis (BNST) to the ventral tegmental area (VTA) midbrain dopamine (DA) neurons, which are required for processing naturally rewarding stimuli as well as self-administration of drugs of abuse. Tract tracing approaches, electron microscopy, and in vivo electrophysiological recordings were used to demonstrate that the BNST actively relays the excitatory drive from the infralimbic cortex (ILCx) to VTA DA neurons, and that intra-BNST CB1-R stimulation inhibits the firing of dopamine cells evoked by stimulation of the ILCx.
CB1R immunocytochemistry relies on the availability of highly specific antibodies. Thus, CB1R-/- knock-out mice were used as controls for antibody specificity. Mice were deeply anesthetized by intraperitoneal injection of a mixture of Nembutal (5 mg/100 g) and urethane (130 mg/100 g body weight). Rats injected with tracers were deeply anesthetized in the same manner. All animals were transcardially perfused with PBS (0.1 M; pH 7.4), then fixed by 500 mL of a fixative of 0.1% glutaraldehyde, 4% formaldehyde, and 0.2% picric acid in phosphate-buffered saline (PBS). Perfusates were used at 4°C. Tissue blocks were extensively rinsed in 0.1 M PBS, pH 7.4. Coronal 50 µm-thick BNST vibrosections were collected in 0.1 M PBS, pH 7.4, at room temperature (RT). Immunolocalization of CB1-R in CB1R-/- knock-out and wild-type mice was achieved by preembedding immunogold labeling. For CTb/CB1-R or PHA-L/CB1-R, floating sections were preincubated in 20% BSA/PBS for 1 hour at RT and incubated with rabbit PHA-L (1:2000 in 1.5% BSA/PBS) or goat CTb polyclonal antibodies (1:20,000 in 1.5% BSA/PBS) for 3 days at 4°C. Each antibody was incubated with polyclonal guinea pig anti-CB1-R antibodies (3 µg/ml in 1.5% BSA/PBS) for 3 days at 4°C. Immunolocalization of PHA-L/CB1-R and CTb/CB1-R was performed by the preembedding immunogold and immunoperoxidase method: CB1-R WAS revealed by treatment with a Nanogold-labeled goat anti-guinea pig IgG (Fab' fragment; 1:100 in 1.5% BSA/PBS), PHA-L by a biotinylated goat anti-rabbit, and CTb by a biotinylated donkey anti-goat IgG (1:200 in 1.5% BSA/PBS). BNST tissue was washed in PBS and processed by a conventional avidinbiotin horseradish peroxidase complex method (ABC Elite, Vector Laboratories). Tissue was washed in PBS overnight at 4°C, then postfixed in 1% glutaraldehyde in PBS for 10 minutes at RT. After washes in double-distilled water, gold particles were enhanced using HQ Silver for 12 minutes in the dark, then washed in 0.1 M PB, pH 7.4. Sections were subsequently preincubated with 0.05% DAB in 0.1 M phosphate buffer (PB) for 5 minutes, incubated by adding 0.01% hydrogen peroxide to the same solution for 5 minutes, then washed overnight in 0.1 M PB at 4°C. Next day, tissue sections were osmicated, dehydrated, and embedded in Epon 812 resin. Ultrathin sections (80 nm) were collected on mesh nickel grids, stained with lead citrate, and examined in the transmission electron microscope.
Tract-tracing experiments showed that BNST neurons that project to the VTA receive potent projections from the infralimbic cortex. A double-labeling strategy combining injection of the anterograde tracer PHA-L in the ILCx and injection of CTb in the VTA confirmed that cortical projections arising from the infralimbic cortex converge on BNST neurons, which in turn project to VTA. The greatest density of CTb-labeled neurons was found in the anteromedial portion of the BNST, coincident with PHA-L-cortical labeling that originated from the ILCx. This shows that ILCx exerts a phasic excitatory influence over the firing characteristics of BNST neurons projecting to the VTA, and provides the first anatomical and functional evidence that the BNST relays the excitatory drive from the ILCx to the midbrain DA neurons. Subcellular distribution of CTb and CB1-R in BNST were examined using preembedding immunoperoxidase and immunogold methods, respectively, for electron microscopy. Immunodeposits of the retrogradely transport CTb were found on dendrites of BNST projecting neurons to VTA apposed to a CB1-R immunopositive synaptic terminal and making an asymmetrical synapse with a CB1-R immunonegative terminal. Together with the other experimental data, this indicates that the ILCx efficiently controls DA neurons in the VTA, via glutamatergic synapses localized within the BNST. Cannabinoids have been revealed to mediate presynaptic inhibition of glutamate release within the ExtA, but their role in the BNST remains elusive. This study provides evidence that CB1-R modulates synaptic excitation in BNST neurons that directly project to the VTA. Together, the data provide a mechanism underlying the effects of cannabinoids on the activity of VTA DA cells.
These findings further recognize the prominent role of the BNST in the control of DA cells excitability and identify a new neuronal substrate for the effects of cannabinoids on reward-related behaviors. The bed nucleus of the stria terminalis (BNST) was shown to efficiently relay cortical excitation to dopamine neurons of the ventral tegmental area (VTA). Anatomical and in vivo electrophysiological evidence demonstrate that excitatory projections arising exclusively from the infralimbic cortex converge on BNST neurons, which in turn project to and excite >80% VTA dopamine cells. At the ultrastructural level, cannabinoid type 1 receptors are detected within the BNST on axon terminals arising from the infralimbic cortex. Intra-BNST infusion of a cannabinoid agonist inhibits the firing of dopamine cells evoked by stimulation of the infralimbic cortex. These data identify a new neuronal substrate for the actions of cannabinoids in the reward pathway.
- Massi, L.; Elezgarai, I.; Puente, N.; Reguero, L.; Grandes, P.; Manzoni, O. J., and Georges, F.: Cannabinoid receptors in the bed nucleus of the stria terminalis control cortical excitation of midbrain dopamine cells in vivo. J. Neurosci., 28, 10496-10508 (2008).
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Are you looking for laboratory work, and interested in helping us develop novel cancer therapeutics based on our gold nanoparticle technology? Currently, we are looking for the following:
Cancer Research Laboratory Assistant
This is an entry level position at Nanoprobes to develop new diagnostic and therapeutic agents for cancer. We are looking for a recent undergraduate college degree in chemistry, biology, or pharmacology and a willingness to work in a novel field with potentially wide applications to cancer diagnosis and therapy.
Nanoprobes is a research and development-based company that develops innovative technology and materials for biomedical research, biological microscopy, medical imaging and nanobiotechnology.
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We have recently described our application of gold-based cancer therapy to the in vivo application of gold nanoparticles. Gold absorbs X-rays more strongly than most tissue, and this causes more dose to be locally deposited at sites where they accumulate. Vascularization of tumors provides a natural mechanism for accumulation of gold nanoparticles of specific sizes, and if a tumor could be specifically loaded with gold, the tumor dose would be increased, and radiotherapy would be enhanced. Our recent article in Physics in Biology and Medicine confirms that this is possible. For more details, see the reference below:
- Hainfeld, J. F.; Slatkin, D. N., and Smilowitz, H. M.: The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol., 49, N309-N315 (2004).
We welcome inquiries from people interested in employment with us, however, please take a few minutes to learn about us and what we do, and how you might fit in, before sending us your resume. Generally, our research and development positions are supported by Small Business Innovation Research (SBIR) grants from the National Institutes of Health and other agencies and have specific project goals, but we are also looking for adaptability and the ability to meet new challenges. Please don't send us more than one copy of your resume - if we are looking, it will get to the right people! For more information, see our Employment page.
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Further demonstration of the utility of Nanogold with gold enhancement, this time for scanning electron microscopy (SEM), was provided by Masyuk and group in their study of the function of Cholangiocyte primary cilia. Gold enhancement is preferred over silver enhancement for SEM because it provides greatly enhanced backscatter detection of the enlarged gold particles, and the authors exploited this fact to localize P2Y in intrahepatic bile ducts (IBDs). Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. The chemosensory function of cholangiocyte cilia was probed by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia, and their involvement in nucleotide-induced cAMP signaling in the cells. Isolated IBDs were immersed in 4% phosphate-buffered glutaraldehyde (pH 7.4) for 1 hour, treated with 0.1% Triton X-100 for 5 minutes, then rinsed with phosphate buffer three times. The samples were incubated overnight at 4°C with anti-P2Y12 antibody (dilution 1 : 100) and then incubated for 2 hours at room temperature with Nanogold anti-rabbit IgG. The samples were fixed in 1% glutaraldehyde for 10 minutes, then gold enhanced for 10 minutes. The samples were dehydrated, critical-point dried, and carbon coated, and images were generated at 8 kV using a Hitachi S-4700 microscope. P2Y12 purinergic receptor was expressed in cholangiocyte cilia: so too are adenylyl cyclases (AC4, AC6, and AC8), protein kinase A (PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150). When ADP, an endogenous agonist of P2Y12 receptors, was perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture, it induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP, but was not affected by UTP. These ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y12 siRNAs, suggesting that cilia and ciliary P2Y12 are involved in nucleotide-induced cAMP signaling. From these results, the authors concluded that cholangiocyte cilia are indeed chemosensory organelles that detect biliary nucleotides through ciliary P2Y12 receptors and transduce corresponding signals into a cAMP response.
A new mechanism for generating image contrast, chemical exchange saturation transfer or CEST, affords an alternative method for magnetic resonance imaging (MRI). Paramagnetic CEST (PARACEST) agents with
chemical exchange groups shifted well away from the bulk water signal offer significant advantages over diamagnetic CEST agents in that faster exchange systems are operable, and in theory each PARACEST active nucleus offers similar contrast to single gadolinium (III) T1 agents; an additional advantage is that the effect can be switched on and off depending on whether a frequency selective presaturation pulse is applied, and the use of alternative nuclei avoids the recently noted nephrotoxic properties of gadolinium. In a recent communication in the Journal of the American Chemical Society, Wu and colleagues describe the preparation of polymeric PARACEST agents which lower the detection threshold for targets by increasing the number of exchanging species at a targeted site. A macrocyclic tetranitrogen donor ligand was polymerized using either 2%, 5%, or 10% (w/w) azo-bis(4-cyanovaleric acid) as initiator in water at 70°C to afford water-soluble, linear polymers differing only in size. After 48 hours, the products were purified by dialysis using a 3 kD MW cutoff membrane, and the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of each polymer were determined by light scattering GPC: the longer species contained up to 18 monomer units. Using Europium (III) derivatives, the detection limits for the longer polymers are in the range 60-80 µM, approaching the levels required for targeted imaging applications.
- Masyuk, A. I.; Gradilone, S. A.; Banales, J. M.; Huang, B. Q.; Masyuk, T. V.; Lee, S. O.; Splinter, P. L.; Stroope, A. J., and Larusso, N. F.: Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors. Am. J. Physiol. Gastrointest. Liver Physiol., 295, G725-G734 (2008).
- Wu, Y.; Zhou, Y.; Ouari, O.; Woods, M.; Zhao, P.; Soesbe, T. C.; Kiefer, G. E, and Sherry, A. D.: Polymeric PARACEST Agents for Enhancing MRI Contrast Sensitivity. J. Amer. Chem. Soc., 130, 13854-13855 (2008).
Gold has therapeutic properties even apart from its ability to enhance radiation therapy, according to Larsen and co-workers in their current Histochemistry and Cell Biology paper. Traumatic brain injury results in loss of neurons, caused as much by the resulting neuroinflammation as by the original injury. Gold salts are known to be immunosuppressive, but their use are limited by nephrotoxicity. However, the authors have previously shown by autometallography that implants of pure metallic gold release gold ions which do not spread in the body, but are taken up by cells near the implant. Therefore, they hypothesized that metallic gold could reduce local neuroinflammation in a safe manner. Bio-liberation, or dissolucytosis, of gold ions from metallic gold surfaces requires the presence of disolycytes i.e. macrophages, and the process is limited by their number and activity. 20-45 µm gold particles were injected into the neocortex of mice before generating a cryo-injury. By comparing gold-treated and untreated cryolesions, it was found that the release of gold reduced microgliosis and neuronal apoptosis, accompanied by a transient astrogliosis and an increased neural stem cell response. It was concluded that bio-liberated gold ions possess pronounced anti-inflammatory and neuron-protective capacities in the brain. This implies that metallic gold has clinical potential. Intra-cerebral application of metallic gold as a pharmaceutical source of gold ions represents a completely new medical concept that bypasses the blood-brain-barrier, and allows direct therapeutic delivery to inflamed brain tissue.
- Larsen, A.; Kolind, K.; Pedersen, D. S.; Doering, P.; Pedersen, M. O.; Danscher, G.; Penkowa, M., and Stoltenberg, M.: Gold ions bio-released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury. Histochem. Cell Biol., 130, 681-692 (2008).
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