letter) who has an established protocol, and agreed to help us on this measurement. The core will collect and process the plasma as instructed and send the sample to Dr. Brosnihan for analysis. We believe that this is the most efficient way to obtain accurate measurements of Ang (1-7) without significant investment. The core will also provide support for the measurement of activities of NOS, SOD, NADPH oxidase and citrate synthase. The core has included Dr. Matthew C. Zimmerman as a co-investigator to help in reactive oxygen species (ROS) analysis because of his specific expertise in this area. The role of NO, Ang II and ROS in controlling cardiovascular dynamics has been adequately documented in all the listed projects. However, the underiying cellular and molecular mechanisms involving these factors are pooriy understood. This core is an integral component of the PPG and will provide service for all projects. Dr. Shaymal K. Roy will be actively involved, as the Director of the core unit, in designing experiments for various protocols, which use RIA, radioreceptor assay, immunohistochemistry. Western immunoblotting, and real time RT-PCR. He will make recommendations and improvements of protocols whenever needed. His expertise in these techniques is critical for the timely completion of the study and analysis of our results. The technical expertise and the equipment involved cannot be reproduced using facilities in any one of the laboratories of the investigators in the Program Project. Because almost all PPG investigators will use these techniques, a core unit will be ideal to meet their needs;thus eliminating major duplication in equipment and manpower. The core has been strengthened by the inclusion of Drs. Zimmerman and Brosnihan to address the cutting-edge questions in the proposed area of research. In fact, during the current funding period, the molecular and biochemical core has helped every investigator listed in the PPG in their research by analyzing samples as well as training post-doctoral fellows, graduate students and technicians of various molecular biology techniques critical for the success of each project. Therefore, Core C has proved its importance in the PPG and has become an indispensable and integral part of the project. The objectives of the present proposal cannot be reached without the assistance of Core C. 249 Zucker, Irving H. Progress Report The use of the Core has increased significantly during the past funding period because of the introduction of several new experimental approaches by investigators. Similar to the previous year, graduate students and post-doctoral trainees utilized the facility to the fullest extent. Because of the increased use of the immunofluorescence histochemistry, the Core has updated the fluorescence microscope with a new Dell computer, metal-halide fluorescence light source and a Leica bright field digital camera. The Core has been doing Western blots, gel-shift assays. Real-time RT-PCR, immunofluorescence, RIA of Ang II, and enzyme immunoassay of noradrenaline. The core has processed over 178 samples for Ang II RIA and 709 samples for NE EIA. In addition, we have processed 458 samples for RT-PCR and 137 samples for Western blots. Besides these, post-doctoral trainees and graduate students have processed their experimental samples themselves using the Core facilities. Altogether, a large number of samples were processed in the Core for various analytical purposes. We expect that the number will go up in future, and additional assays may need to be included as the research interests diversify. Personel in the Core have been using the real-time PCR on a regular basis. The use of commercial non-radioactive gel-shift kits for transcription factor analysis is being continued. Western blot analysis of various phosphorylated proteins has also been introduced in the Core. The Core processed brains of rabbits, mice and rats, rat muscle and carotid bodies, rabbit kidneys and neuronal cell lines during the previous funded period. We have processed dog, rabbit and rat plasma, rat urine and rabbit kidney and brain for norepinephrine analysis. Western blot analysis, gel shift and RT-PCR were used for angiotensin receptor and transcription factor analysis. The Core is working on establishing a chromatin immunoprecipitation (ChIP) assay to examine the transcriptional regulation of angiotensin receptors in tissues and cultured cells (see below). Western blot and RT-PCR analysis were also used for ACE and ACE2 protein and mRNA expression in rabbit brain tissues, ATiR, AT2R eNOS, nNOS, ETaR, ETbR and GAPDH proteins in rabbit kidneys, Gp91, nNOS, and eNOS proteins in rat muscle, ATiR, eNOS, nNOS, NMDA (Glutamate Receptor subtypes), and AMPA proteins in rat brain stem. We are analyzing NFkB phosphorylation using immunoblotting as well as IkB and IkK. As usual, the Core continuously trains post-doctoral and pre-doctoral students, and technicians involved in the PPG. Therefore, the core service is fulfilling its goal. The use of the fluorescence microscope and gel documentation system has again surpassed saturation because investigators are doing more studies that require these techniques. Consequently, we need to upgrade and service the equipment on a regular basis. Our future projection includes immunoblot analysis of Elk-1, Ikk, p38, ERK, PI3kinase and JNK proteins, and gel shift analysis of AP-1 complex. Besides, we will also do Real-time RT-PCR quantification of ATiR, NFkB/p65, and Elk-1 mRNA. The Core budget has been used to procure RT-PCR enzymes, primers and probes for real-time RT? PCR, antibodies, fluorophor-conjugated second antibodies, chemiluminescence substrate, and fine chemicals. The core purchased a small apparatus for electrophoresis and gel processing. The cost of repair and new purchase of pipettes will again be a part of next year's core budget. Overall, the molecular Core C is fulfilling its obligation to all members of the PPG and functions as an integral part of this program. Specific Methodologies A. Immunohistochemistry facility 1. Function This section of the Core provides technical and scientific support for investigators who need to use immunofluorescence histochemistry for localizing specific proteins in cells/tissues, which include neurons, brain sections, carotid bodies, etc. The Core has a Cryostat and paraffin sectioning facilities, and a research microscope equipped with an Q-imaging digital camera for incident-light image capture, a low-light sensitive Q- imaging Retiga fast 1294 camera for fluorescence image capture and appropriate software for image capture and analysis. The Core also provides appropriate secondary antibodies-flurophor conjugates for signal detection. 250 Zucker, Irving H. 2. Services available Mrs. Phyllis Anding, who has the expertise in immunohistochemistry and microscopic imaging, will carry out the procedures. She is certified in radioactive handling and environmental health. Her inclusion in the Core is critical to provide necessary services to the PPG investigators. She will also assist members of various investigators'laboratories in the above techniques. Dihydroethidium staining In order to evaluate the level of ROS in brain, muscle and carotid bodies, we will stain for the ROS marker Dihydroethidium (DHE). Unfixed fresh frozen sections (30 pm in thickness) will then be incubated with DHE (1x10""""""""? M, Sigma, St. Louis, MO) and Hoechst 33258 (2x10'^ M, nuclear stain. Molecular Probes Inc., Eugene, OR), in PBS for 15 minutes in the dark at room temperature. Sections will be rinsed 3 times in PBS, coversliped and evaluated under epifluorescence in a Leica DMR research microscope equipped with an Optronics Magnafire digital camera (Optronics, Goleta, CA) and digitally photographed. Dr. Zimmerman will be consulted as necessary. General Immunofluorescence histochemistry protocol^ For all immunofluorescence histochemistry, tissues will be snap frozen in liquid nitrogen (LN2) and brought to cryostat temperature at -15?C and sectioned at 10 pm thickness. Upon thawing on a slide warmer at 40?C the sections will be fixed in 2% freshly prepared paraformaldehyde in PBS, pH 7.4 at 4?C, rinsed thoroughly in PBS, and non-specific IgG binding sites blocked with 10% donkey serum in PBS containing 0.05% Tween 20 for 1 h at 4?C. After removing the blocking solution, sections will be incubated overnight with antigen-specific primary antibodies at 4?C, rinsed with PBS and the signal will be generated by incubating with the corresponding host-specific second antibody conjugated to Alexa 488 (green fluorescence), or Alexa 596 (red fluorescence) for uni- or multi-labeling. Nuclei will be stained with DAPI (blue) depending on the color of the antigen signal and viewed under an epifluorescence microscope. Periodically we will also use confocal microscopy for detail analysis of particular sections for specific experiments. UNMC imaging core facility contains two Zeiss multi-laser confocal microscopes, and service Is available for $40/h. For digital microphotography, the exposure time will be set using control sections, which have been incubated with either non-immune IgG or a particular primary antibody that has been preneutralized with 100-fold excess antigen, to eliminate any background fluorescence. The signal obtained after background correction will be considered specific and recorded digitally using a Q-imaging Retiga 1394 digital camera. In some instances, the intensity of the specific fluorescence will be quantified using NIH Image software. Immunohistochemistry protocols mentioned above will be carried out using antigen specific antibodies. For some proteins we will use antibodies, which have multispecies or monospecies specificity because the detection will involve endogenous enzyme proteins as well as proteins expressed by viral transgene. Monospecies specific antibody is particulariy important because it will allow us to detect (either by immunofluorescence or Western blotting) selectively human protein produced by the transgene at the injected areas without any contribution of the rat or rabbit protein. We have optimized the signal detection using these antibodies and been using during the previous funding period. Anticipated results, pitfalls and alternative approaches. Based on the experience during the current funding period, no major problems are anticipated. Unavailability of the pure antigens for immunoneutralization of the primary antibodies often presents a limitation to verify the specificity of the immunostaining. Although antibody host-specific IgG and no primary antibody controls sometime provide acceptable data, they are not always useful because the primary antibody, but not the preimmune IgG of the same species, often produce false positive staining. To circumvent this problem, we used tissues wherein some cells serve as negative control because they do not express the antigen of interest, thus helping us to judge the specificity of the staining. Second, we always verify the specificity of the antibody using Western blot analysis using positive and negative controls. 251 Zucker, Irving H. B. Enzyme assay, and protein electrophoresis and immunoblotting facility 1. Function This section of the core provides technical and scientific support for investigators who need to use enzyme photochemical and biochemical assays to detect NOS activity and protein levels, levels of ecNOS, ATiR and SOD proteins. The laboratory has a cryostat, incubator, microscopy and imaging facility, water bath, spectrophotometer for quantifying protein content, a complete system for polyacrylamide gel electrophoresis and Western immunoblotting, and UVP Bioimagerfor chemiluminescence signal detection. 2. Services available Mrs. Phyllis Anding will carry out these procedures. Mrs. Anding has been trained to perform these assays during the current funding period and has proven to be an essential member of the core. Without her help, timely service cannot be provided. The technologist will be adept at performing biochemical assay protocols as well as assisting members of various investigators'laboratories in the above techniques. SOD activitv assay SOD activitv: In-gel assay CuZnSOD and MnSOD activity will be measured using a native-gel activity stain as previously described^'^. After separating cell lysates on a native gel using electrophoresis, SOD enzyme activity bands will be visualized by saturating the gel with 2.6 mM nitroblue tetrazolium (NBT), 28 mM riboflavin, and 28 mM tetramethylethylenediamine (TEMED) for 20 min in the dark and then illuminated under a fluorescent light until achromatic bands appeared. SOD activitv: Spitz/Oberlev spec assay'' Briefly, brains will be rapidly removed, flash frozen in dry ice, and sectioned to the anterior border of the lateral ventricles. Tissue surrounding the ventricles, including the lamina terminalis and SFO, will be dissected using a micropunch (1.24 mm diameter) and homogenized on ice with a Teflon pestle doused in 0.05 mol/L phosphate buffer (pH 7.8) with 1 mmol/L diethylenetriamine-pentaacetic acid (DETAPAC). Tissue protein content will be measured by the method of Lowry et al.""""""""* and increasing quantities (0 - 500|ig) will be placed in a solution containing 1 mmol/L DETAPAC, 1 unit of catalase, 5.6x10'^ mol/L nitro blue tetrazolium (NBT), 10""""""""^ mol/L xanthine, 5 mmol/L NaCN, and 50 |xmol/L bathocuproine disulfonic acid. Xanthine oxidase will be added to initiate superoxide-mediated NBT reduction to blue formazan, and the rate of formation of colored product will be monitored spectrophotometrically at 560 nm^. The rate of NBT reduction in the absence of tissue will be used as the reference rate. Data will be expressed in units of SOD activity per milligram protein, where 1 unit is defined as the amount of enzyme needed to cause a 50% reduction in product formation using purified CuZnSOD in the absence of ON'as the standard^. NADPH Oxidase assay This will be done essentially as described by Ushio-Fukai et al.? Briefly, areas of brain from rats and rabbits, and carotid bodies from rabbits will be dissected, snap frozen in LN2, and homogenized in 0.05 M KPO4 buffer, pH 7.4 containing 10% glycerol and a protease inhibitor cocktail in a teflon-Dounce homogenizer. After measuring the protein content by micro BCA protein assay kit, the enzyme activity will be measured in a luninescence assay with 500 mM lucigenin as the electron acceptor and 100 mM NADPH as the substrate in a final volume of 0.9 ml. The reaction will be started by adding 100 ml of homogenate containing 50 mg protein, and photon emission will be measured every 15 s for 10-15 min in a luminometer available in the department of Biochemistry and Molecular biology at UNMC (7*^ floor of DRC building). The initial rate of enzyme activity will be calculated by linear regression. A buffer blank (<5% of the cell signal) will be subtracted from each reading prior to transformation of the data to nanomoles of O2, using a standard curve generated with xanthine/xanthine oxidase. Activity in a subset of samples will be measured in the presence of Tiron (20 mmol/l), a superoxide anion scavenger, to confirm that the signal is attributable to superoxide anion, and in the presence of diphenylene-iodonium (DPI, 10 jxmol/l), an NAD(P)H oxidase inhibitor, to confirm the activity is attributable to the enzyme. Citrate synthase assay This will be done according to the method described by Srere^ with slight modification. Breifly, tissues will be homogenized in 0.4 M KCI containing 10% glycerol, pH 7.4, and centrifuged at 23,000g for 30 min at 252 Zucker, Irving H. 4?C. The supernatant will be separated, protein concentration determined and 5 ml of sample containing 20-30 mg protein mixed with 945 ml of a reaction mixture containing (final concentration) 0.1 mM dithiobis-nitro- benzoate (DTNB), 0.3 mM acetyl coA in deionized water. After taking readings at 412 nm for 3 min at 1 min intervals without the substrate, 50 ml of a 10 mM oxaloacetate will be added and the absorption will be followed for 3 more minutes at 1 min intervals at 37?C. Enzyme activity will be expressed as nmol mercaptide formed/min/ mg protein. Western Immunoblotting Commercially available antigen specific mono or polyclonal antibodies will be used. Briefly, protein samples will be prepared by homogenizing tissues in 1x RIPA [PBS containing 1% NP-40, 0.5% deoxycholate and 0.1% SDS, pH 7.4]. The concentrations of protein in the samples will be determined by a Micro BCA protein assay kit (Pierce Chemical Company), and equal amounts of protein from all samples will be resolved in 10% polyacrylamide gel, transferred to PVDF membrane, blocked with 5% milk in Tris-buffered saline, pH 7.0 containing 0.05% Tween 20, and probed with antigen specific antibody. The signal will be generated using corresponding host-specific second antibody-peroxidase conjugate and WestFemto chemiluminescence substrate (Pierce). The light emanating from the specific protein band will be directly digitized using an UVP bioimager. The data will be presented as OD per mg protein. The same membrane will also be evaluated for b- tubulin protein to determine the specificity of changes in the levels of antigens, and for correcting any variability due to differences in sample loading. Electrophoretic Mobility Shift Assay (EMSA) ATiR expression is upregulated by the AP-1 and NFkB complexes. To determine the molecular mechanisms underiying the ATiR gene expression, we will examine the binding activity of transcription factors present in tissue samples using synthetic transcription factor consensus binding sites in EMSA. Two complementary oligos will be synthesized in the UNMC Eppley DNA Synthesis Core Facility at the University of Nebraska Medical Center. The oligomers will be labeled at the 3'end with biotin using the 3'End DNA Labeling Kit (Pierce), then annealed at room temperature to obtain double-stranded DNA. The binding reactions will contain 10 pg of nuclear extract protein, buffer (10 mM Tris, pH 7.5, 50 mM KCI, 5 mM MgCI2, 1 mM dithiothreitol, 0.05% Nonidet P-40, and 2.5% glycerol), 1 pg of poly(dl-dC), and 2 nM of biotin-labeled DNA. The reactions will be incubated at 23 """"""""C for 20 min. The competition reactions will be performed by adding 100-fold excess unlabeled double stranded AP-1 or NFkB consensus oligonucleotides to the reaction mixture. In antibody supershift assays, incubation will be performed in the absence or presence of 2 pg of anti- c-Jun antibody (Santa Cruz Biotechnology) added to the reaction mixture. The reactions will then be electrophoresed on a 5% native polyacrylamide gel in 0.5X TBE at 100 V for 1 h in 0.5X TBE buffer. The reactions will be transferred to a nylon membrane. The biotin-labeled DNA will be detected with a LightShift chemiluminescent electrophoretic mobility shift assay kit (Pierce). The bands on the membrane will be digitized and analyzed using UVP Biolmaging Systems. We have used this assay in previous experiments as indicated in Project 1?'^. Similar protocols will be used for other transcription factors. Chromatin Immunoprecipitation (ChIP) Assay In order to determine precise binding of a give transcription factor to the promoter regions of specific genes we will use a ChIP assay. Our test material will include Cath.a and NG-108 cells as well as tissue punch biopsies take from various brain areas such as the RVLM, NTS and PVN in our animal models (rat, mouse, rabbit). ChIP would be done after appropriate experimental protocols, using the ChIP assay kit from Upstate (Lake Placid, NY) as per the manufacturer's instructions. Briefly, after appropriate treatment, cells will be washed in cold PBS and crosslinked by adding formaldehyde to a final concentration of 1%. Tissue samples will first be homogenized and then treated with formaldehyde. Nuclear extraction after formaldehyde fixation would be done in lysis buffer (50 mM Tris [pH 8,0], 2 mM EDTA, 0.1% Nonidet P-40, and 10% glycerol) supplemented with protease inhibitors. Chromatin will be sheared by sonication, centrifuged, and pre-cleared for 3 h with salmon sperm-saturated protein A/G- agarose (ssProtein A/G). Immunoprecipitation would be carried out overnight at 4?C using 5 pi of anti- p65 253 Zucker, Irving H. antibody (Cell Signaling) and the immune complexes collected with ssProtein A/G for 30 min and washed three times in washing buffer and once in 0.5 M LiCI, followed by three washes with TE buffer. Immune complexes would be extracted 3 times with 100 pi of extraction buffer and DNA cross-links reverted by heating for 8 h at 65?C. After proteinase K (100 pg for 2 h) digestion, DNA would be extracted with phenol/chloroform and precipitated in ethanol. PCR would be done with total DNA (1 pi, input control) and immunoprecipitated DNA (2 pi) using the appropriate species-specific and promoter-specific primers. C. RNA preparation and RT-PCR facility 1. Function This section of the Core provides technical and scientific support for investigators who will quantify the relative steady-state levels of mRNA in tissues. The Core laboratory has a microfuge, a thermocycler, water baths, areas for RNA extraction, agarose gel electrophoresis, capillary blotting. Southern and Northern hybridization oven, and a dedicated area for RT-PCR amplification of mRNA. We also have a MJ Research Opticon 4 thermocycler for real time quantification of mRNA levels. 2. Service available The overall function of the facility will be supervised by Dr. Shaymal K. Roy, who will make recommendations about relevant protocols to suit the investigators'needs. Phyllis Anding will be responsible for RNA extraction and real-time RT-PCR evaluation of various gene transcripts. Mrs. Anding has been fully involved in this capacity during the current funding period, and is well trained to undertake the task. Her inclusion in the Core is essential for timely progress of the study. She will also assist members of various investigators'laboratories in the above techniques. Extraction of RNA from tissue samples Total RNA will be extracted from various areas of the rat and rabbit and mouse brains, and rabbit carotid bodies using RNAeasy mini kit (Qiagen, CA) according to the manufacturer's protocol. In brief, tissues frozen in liquid N2 will be thawed directly in the lysis buffer at room temperature and homogenized using an Omni homogenizer. After 5 min centrifugation to remove undissolved debris, the supernatant will be mixed with 70% ethanol and loaded on to RNA binding columns. The columns will be centrifuged, washed with appropriate buffer and the RNA eluted in small volume of RNase-free water, and quantified spectrophotometrically at 260/280 nm. The RNAeasy protocol is based on chaotropic salt, which allows very clean RNA preparation in a very short time and without generating any phenol by-product. Because the core has been processing an enormous number of samples for RNA preparation, using method that can be done quickly without any compromise in the RNA quality or yield is essential. Quantification of NOS. NADPH oxidase. SOD and AT
Showing the most recent 10 out of 169 publications
