Scott Henderson
Scripps Research Institute, Molecular Medicine, Faculty Member
- https://www.scripps.edu/faculty/henderson/
http://www.researchgate.net/profile/Scott_Henderson
http://www.linkedin.com/pub/scott-henderson/13/281/9b3edit
Background: Right ventricular (RV) ischemia occurs in pulmonary hypertension (PH) patients despite normal coronary angiograms. O 2 supply-demand balance in the pressure-overloaded RV is determined by the interplay between reduced coronary... more
Background: Right ventricular (RV) ischemia occurs in pulmonary hypertension (PH) patients despite normal coronary angiograms. O 2 supply-demand balance in the pressure-overloaded RV is determined by the interplay between reduced coronary perfusion (due to systemic hypotension and compression of coronary vessels) and a wall stress-related increase in myocardial O 2 consumption. The role of capillary rarefaction, which is involved in pressure overload induced left heart failure, has not been investigated in PH-associated RV failure. Methods: Rats received a single dose of the VEGF receptor blocker SU5416 and were exposed to 4 wks of normobaric hypoxia and 2 wks of sea level room air. This regimen induces angioproliferative changes in the pulmonary circulation, severe PH and RV failure. At 6 wks, the rats received intravital injections of Texas-Red conjugated tomato lectin, which is an N-acetylglucosamine specific marker of blood vessels in rodents. After circulation of the lectin for 5 min, papaverin was injected to promote maximal vasodilation of all perfused blood vessels. Animals were exsanguinated and perfusion-fixed with paraformaldehyde. After overnight fixation, 300 μm thick transverse sections of the myocardium were bleached in a methanol/DMSO mixture and nuclei were stained using DAPI. Prior to 3-dimensional imaging under a confocal microscope, further tissue clearing was achieved using organic solvents. Results: Z-stacks of 2-dimensional images were made in whole-mount cardiac specimens, using a step size of 0.32 μm. 3-dimensional reconstruction allowed quantification of the total capillary volume, which was reduced in the PH model compared to controls (2.9±1% vs. 5.7±1% of tissue volume, p Conclusions: RV failure in experimental PH is associated with capillary rarefaction. 3-dimensional imaging of the myocardial microcirculation can be achieved using intravital injections of tomato lectin and subsequent confocal imaging.
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&NA; H460 non‐small cell lung, HCT116 colon and 4T1 breast tumor cell lines induced into senescence by exposure to either etoposide or doxorubicin were able to recover proliferative capacity both in mass culture and when enriched for... more
&NA; H460 non‐small cell lung, HCT116 colon and 4T1 breast tumor cell lines induced into senescence by exposure to either etoposide or doxorubicin were able to recover proliferative capacity both in mass culture and when enriched for the senescence‐like phenotype by flow cytometry (based on &bgr;‐galactosidase staining and cell size, and a senescence‐associated reporter, BTG1‐RFP). Recovery was further established using both real‐time microscopy and High‐Speed Live‐Cell Interferometry (HSLCI) and was shown to be accompanied by the attenuation of the Senescence‐Associated Secretory Phenotype (SASP). Cells enriched for the senescence‐like phenotype were also capable of forming tumors when implanted in both immunodeficient and immunocompetent mice. As chemotherapy‐induced senescence has been identified in patient tumors, our results suggest that certain senescence‐like phenotypes may not reflect a terminal state of growth arrest, as cells that recover with self‐renewal capacity may ultimately contribute to disease recurrence.
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Significance Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes which entered the host cell and were retained as endosymbionts. Here, we model this earliest stage of the endosymbiotic theory of... more
Significance Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes which entered the host cell and were retained as endosymbionts. Here, we model this earliest stage of the endosymbiotic theory of mitochondrial evolution by engineering endosymbiosis between two genetically tractable model organisms, Escherichia coli and Saccharomyces cerevisiae . In this model system, we engineered E. coli strains to survive in the yeast cytosol and provide ATP to a respiration-deficient yeast mutant. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. This readily manipulated chimeric system was stable for more than 40 doublings and should allow us to investigate various aspects of the endosymbiotic theory of mitochondrial evolution.
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Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their... more
Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets...
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Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These... more
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and i...
Research Interests: Biology, Immunohistochemistry, Medicine, Multidisciplinary, Mammals, and 15 moreElectricity, Cerebral Cortex, Animals, Astrocyte, Central Nervous System, Astrocytes, PLoS one, Nerve Regeneration, Rats, Intensity, Glial Fibrillary Acidic Protein, CNS, Cell Proliferation, Glial scar, and Electric stimulation
Autophagy is a conservative biological process, which involves the degradation of intracellular components (proteins or organelles) via the fusion of autophagosomes with lysosomes. Autophagy has been reported to have both cytoprotective... more
Autophagy is a conservative biological process, which involves the degradation of intracellular components (proteins or organelles) via the fusion of autophagosomes with lysosomes. Autophagy has been reported to have both cytoprotective and cytotoxic actions in response to either chemotherapy or radiation in different tumor model systems. In previous studies (Elmore LW et al., J Biol Chem. 2002 Sep 20; 277(38): 35509-15.), Adriamycin (doxorubicin), one of the primary chemotherapeutic drugs utilized in the treatment of breast cancer, was shown to induce a pronounced senescence response within 72hrs in MCF-7 breast tumor cells (at a clinically relevant concentration of 1μM, with a 2 hr exposure). Interestingly, Adriamycin was also observed to cause a time-dependent increase of autophagy within 72hrs (by acridine orange, Monodansylcadaverine) staining and electron microscopy). The current study was designed to evaluate the relationship between Adriamycin-induced autophagy and senescenc...
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Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the ‘‘9+2’’ axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair... more
Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the ‘‘9+2’’ axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L an...
Research Interests: Biology, Medicine, Gene expression, Multidisciplinary, Protein Structure and Function, and 15 moreCell line, Humans, Germ Cells, Spermatogenesis, Mice, Animals, Male, tESTIS, PLoS one, mRna expression levels, Protein isoforms, Ribonucleoproteins, Chlamydomonas Reinhardtii, Germ Cell, and Nuclear Localization
Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their... more
Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macro-phage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor a underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These... more
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues , as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair.
Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates.... more
Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates. Because SPAG6 decorates microtubules, we hypothesized that SPAG6 has other roles related to microtubule function besides regulating flagellar/cilia motility. Mouse embryonic fibroblasts (MEFs) were isolated from Spag6-deficient and wild-type embryos for these studies. Both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFs, and they had a larger surface area. Re-expression of SPAG6 in the Spag6-deficient MEFs rescued the abnormal cell morphology. Spag6-deficient MEFs were less motile than wild-type MEFs, as shown by both chemotactic analysis and wound-healing assays. Spag6-deficient MEFs also showed reduced adhesion associated with a non-polarized F-actin distribution. Multiple centrosomes were obser...
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Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed... more
Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.
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Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract... more
Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen-17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with "9 + 2" motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.
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Fluorescence labeled oligonucleotides have a long history of being used to monitor nucleic acid transport and uptake. However, it is not known if the fluorescent moiety itself physically limits the number of pathways that can be used by... more
Fluorescence labeled oligonucleotides have a long history of being used to monitor nucleic acid transport and uptake. However, it is not known if the fluorescent moiety itself physically limits the number of pathways that can be used by the cell due to steric, hydrophobic, or other chemical characteristics. Here, we report a method for comparing the uptake kinetics of oligonucleotides labeled either with the fluorescent pteridine, 3-methyl-8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI), or the common fluorophore 5-carboxyfluorescein (5-FAM). We use a multiphoton microscopic technique to monitor nucleic acid uptake LLC-PK1, a pig renal tubular cell line that is known to have multiple uptake pathways. We find that the two fluorophores enter the cells at different rates, suggesting that choice of fluorescent moiety influences the uptake pathway used by a cell. Finally, we reconstituted an LLC-PK1 membrane channel that is selective for nucleic acids in planar lipid bilayers, and tested the ability of the labeled nucleic acids to permeate the channel. We find that 3MI, and not 5-FAM labeled oligonucleotides can traverse the plasma membrane through the channel. These results have implications for future studies aimed at delivering pteridine moieties to cells and for tracking nucleic acid transport into tissues.
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Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract... more
Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen-17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with "9 + 2" motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.
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Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was... more
Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line. Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on β-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays. Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy. Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.
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Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations. To prevent this trauma from catalyzing genomic... more
Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1. The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones, forming foci within 5-15 minutes. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis. In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.
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Glioblastoma multiforme is the most common and most lethal primary brain tumor in humans, with median survival of approximately 1 year. Owing to the ability of glioma cells to aggressively infiltrate normal brain tissue and survive... more
Glioblastoma multiforme is the most common and most lethal primary brain tumor in humans, with median survival of approximately 1 year. Owing to the ability of glioma cells to aggressively infiltrate normal brain tissue and survive exposure to current adjuvant therapies, there is a great need for specific targeted nanoplatforms capable of delivering both therapeutic and imaging agents directly to invasive tumor cells. Gadolinium-containing endohedral fullerenes, highly efficient contrast agents for MRI, were functionalized and conjugated with a tumor-specific peptide and assessed for their ability to bind to glioma cells in vitro. We report the successful conjugation of the carboxyl functionalized metallofullerene Gd(3)N@C(80)(OH)(-26)(CH(2)CH(2)COOH)(-16) to IL-13 peptides and the successful targeting ability towards brain tumor cells that overexpress the IL-13 receptor (IL-13Rα2). These studies demonstrate that IL-13 peptide-conjugated gadolinium metallofullerenes could serve as a platform to deliver imaging and therapeutic agents to tumor cells.
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Research Interests: Fluorescence Resonance Energy Transfer, Signal Transduction, Cell and Molecular Biology, Biological Sciences, RNA interference, and 12 moreCell line, Humans, Endothelial Cells, Mutation, Molecular Cell Biology, Time Factors, Signaling, Transfection, Protein Conformation, Structure activity Relationship, Ligands, and Cell Membrane
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Research Interests: Neuroscience, Psychology, Gene expression, Mammals, PCR, and 19 moreHippocampus, Electroporation, Animals, Asic, Green Fluorescent Protein, Single Cell, Rats, Sodium channel, Voltage-Gated Sodium Channels, Fluorescent Protein, Action potential, Species Specificity, Action Potentials, Heterologous Expression, P, GFP, Neurosciences, FMRFamide, and Persistent Excitation
Research Interests: Spine, Brain, Dendritic Spines, Models, Animals, and 16 moreDendrites, Phosphorylation, Immunoprecipitation, Epilepsia, Clinical Sciences, Rats, Pilocarpine, Analysis of Variance, Time Factors, Cognitive Function, Cholinergic Receptor, Calcineurin, Status Epilepticus, Subcellular Fractions, Muscarinic Receptor, and Neurosciences
Research Interests: Materials Engineering, Chemical Engineering, Image Processing, Tissue Engineering, Scanning Electron Microscopy, and 9 moreAnisotropy, Fourier Analysis, Spatial Information, Spatial Organization, Structural Properties, Fast Fourier Transform, Biocompatible Materials, Frequency Domain, and Mechanical Loading
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Gbeta5 exists as two splice variants, Gbeta5-S and Gbeta5-L, which interact with and stabilize the R7 members of the regulators of G-protein signaling (RGSs): RGS6, RGS7, RGS9, and RGS11. Although the role of Gbeta5-L and RGS9-1 is... more
Gbeta5 exists as two splice variants, Gbeta5-S and Gbeta5-L, which interact with and stabilize the R7 members of the regulators of G-protein signaling (RGSs): RGS6, RGS7, RGS9, and RGS11. Although the role of Gbeta5-L and RGS9-1 is established in photoreceptors, the physiological functions of Gbeta5-S and other R7 RGS proteins remain unclear. We found that the electroretinogram of Gbeta5-/- mice lacks the b-wave component and that Gbeta5-S and RGS11 colocalize with Go alpha at the tips of the ON-bipolar cell dendrites. Unexpectedly, we found a significant reduction in the number of synaptic triads in the outer plexiform layer (OPL) of the Gbeta5-/- mice, which is evident at postnatal day 14. Transgenic expression of Gbeta5-L in rods failed to rescue the b-wave or the OPL defects. These results indicate that Gbeta5-S is indispensable for OPL integrity and normal light responses of the retina.
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Research Interests: Cognitive Science, Fluorescence Microscopy, Transmission Electron Microscopy, Fluorescent Dyes and Reagents, Confocal Microscopy, and 14 moreBrain, Animals, Reaction Time, Neurons, Astrocytes, Tunable Filter, Rats, Glial Fibrillary Acidic Protein, Confocal Laser Scanning Microscopy, Cellular Structure, Statistical Methods for Neuroscience, Neurosciences, Low Resolution, and Region of Interest
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Research Interests: Neuroscience, Psychology, Working Memory, Image Analysis, Scanning Electron Microscopy, and 34 moreConfocal Microscopy, Surface Roughness, Hippocampus, Dendritic Spines, Humans, Image Reconstruction, Mice, Female, Animals, Male, Three Dimensional Imaging, Neurons, Shape Analysis, Green Fluorescent Protein, Diagnostic Imaging, Structural Change, Amyloid Beta, Aged, High resolution satelite image, Cognitive impairment, Cross Section, Three Dimensional, Neurodegenerative Disease, Confocal Laser Scanning Microscopy, Complex Structure, Alzheimer Disease, Laser Scanning Microscopy, D, Neurosciences, Numerical Aperture, Normal Aging, CLSM, Cell Size, and Ad
Genetic diagnosis of preimplantation embryos (PGD) can substantially reduce the chance that at-risk couples have children afflicted with inherited diseases. However, PGD requires DNA,which is usually obtained from single cells following... more
Genetic diagnosis of preimplantation embryos (PGD) can substantially reduce the chance that at-risk couples have children afflicted with inherited diseases. However, PGD requires DNA,which is usually obtained from single cells following embryo biopsy. In addition, PGD requires that the genetic defect(s) causing the disorder be known. We have therefore developed an alternative to PGD, which we term preimplantation enzymatic diagnosis (PED). PED has several advantages over PGD, including the facts that it does not require embryo biopsy and that the gene defect(s) causing the disorder need not be known. We have demonstrated 'proof of principle' for this approach using embryos obtained from a mouse model (ASMKO mice) of acid sphingomyelinase (ASM)-deficient Niemann-Pick disease, an inherited lysosomal storage disorder. For this technique, fluorescently (BODIPY)-conjugated sphingomyelin was used to detect ASM activity in situ. Wild-type, preimplantation embryos degraded the substrate following a short 'pulse-chase' period, resulting in markedly reduced fluorescence compared to ASMKO embryos, which retained the fluorescent substrate. Thus, the two embryo types could be easily distinguished by fluorescent microscopy. The fluorescent sphingomyelin was not toxic to the embryos, and the entire procedure could be accomplished within 48 h without embryo biopsy. We suggest that PED may be useful for the preimplantation diagnosis of lysosomal storage disorders, and perhaps other enzymatic defects where similar in situ assay methods are available.