Contemporary operative approaches to the internal auditory canal (IAC) require the creation of large surgical portals for visualization with associated morbidity, including hearing loss, vestibular dysfunction, facial nerve injury, and... more
Contemporary operative approaches to the internal auditory canal (IAC) require the creation of large surgical portals for visualization with associated morbidity, including hearing loss, vestibular dysfunction, facial nerve injury, and skull base defects that increase the risk of cerebrospinal fluid leak. Transcanal approaches to the IAC have been possible only via a transcochlear technique. To preserve cochlear function, we describe a novel endoscopic transcanal infracochlear approach to the IAC in cadaveric temporal bones. Navigation fiducials were secured on fresh cadaveric heads, and real-time computed tomography imaging was used for surgical guidance. With a combination of curved instruments and rigid angled endoscopy, a transcanal hypotympanotomy and subcochlear tunnel were created with superior extension to access the IAC. Postprocedure imaging and temporal bone dissection confirmed access to the IAC without injury to the cochlea or neighboring neurovascular structures.
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Sensory organ damage induces a host of cellular and physiological changes in the periphery and the brain. Here, we show that some aspects of auditory processing recover after profound cochlear denervation due to a progressive,... more
Sensory organ damage induces a host of cellular and physiological changes in the periphery and the brain. Here, we show that some aspects of auditory processing recover after profound cochlear denervation due to a progressive, compensatory plasticity at higher stages of the central auditory pathway. Lesioning >95% of cochlear nerve afferent synapses, while sparing hair cells, in adult mice virtually eliminated the auditory brainstem response and acoustic startle reflex, yet tone detection behavior was nearly normal. As sound-evoked responses from the auditory nerve grew progressively weaker following denervation, sound-evoked activity in the cortex-and, to a lesser extent, the midbrain-rebounded or surpassed control levels. Increased central gain supported the recovery of rudimentary sound features encoded by firing rate, but not features encoded by precise spike timing such as modulated noise or speech. These findings underscore the importance of central plasticity in the perceptual sequelae of cochlear hearing impairment. VIDEO ABSTRACT.
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Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and... more
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly…
Research Interests: Transcription Factors, Gene expression, Multidisciplinary, Nature, Single molecule, and 16 moreTranscriptome, Cell line, Humans, Mice, Human Body, Animals, Cluster Analysis, Spatial Organization, Genome, Gene Regulatory Networks, Transcription Factor, Expression analysis, Binding Site, Transcription Start Site, Gene Expression Regulation, and Functional Annotation
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To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. Primary pigmented RPE... more
To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. Primary pigmented RPE harvested from third trimester fetal pigs were transplanted as microaggregates into the subretinal space of 3 albino rabbits. We then used an in situ probe for a repetitive segment of the porcine chromosome to identify the transplanted RPE. Pigmented cells were visible in the subretinal space 2 weeks after transplantation. Approximately 70% of pigment-containing cells were also labeled with the porcine chromosomal marker. Labeled cells were predominantly flatter in morphology and close to Bruch's membrane whereas unlabeled cells were rounder and further from Bruch's membrane. The outer nuclear layer thickness was normal above the pigmented monolayer but was decreased over areas containing multiple layers of pigmented cells. Fetal porcine RPE xenografts can be identified with a nuclear marker for a repetitive segment of the porcine chromosome. The presence of pigment within unlabelled cells suggests that pigment is not a robust marker for transplanted RPE.
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HMG domain transcription factor, Sox2, is a critical gene for the development of cochlear hair cells, the receptor cells for hearing, but this has been ascribed to expansion of the progenitors that become hair cells. Here, we show that... more
HMG domain transcription factor, Sox2, is a critical gene for the development of cochlear hair cells, the receptor cells for hearing, but this has been ascribed to expansion of the progenitors that become hair cells. Here, we show that Sox2 activated Atoh1, a transcription factor important for hair cell differentiation, through an interaction with the 3' enhancer of Atoh1. Binding to consensus sequences in the Atoh1 enhancer was dependent on the level of Sox2, and the extent of enhancer binding correlated to the extent of activation. Atoh1 activation by Sox2 was required for embryonic hair cell development: deletion of Sox2 in an inducible mutant, even after progenitor cells were fully established, halted development of hair cells, and silencing also inhibited postnatal differentiation of hair cells induced by inhibition of γ-secretase. Sox2 is thus required in the cochlea to both expand the progenitor cells and initiate their differentiation to hair cells.
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In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria... more
In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points. We identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity. ...
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The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of... more
The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of regenerative capacity. Recent studies revealed the existence of stem cells in the postnatal spiral ganglion, which gives rise to the hope that these cells might be useful for regenerative inner ear therapies. Here, we provide an in-depth analysis of sphere-forming stem cells isolated from the spiral ganglion of postnatal mice. We show that spiral ganglion spheres have characteristics similar to neurospheres isolated from the brain. Importantly, spiral ganglion sphere cells maintain their major stem cell characteristics after repeated propagation, which enables the culture of spheres for an extended period of time. In this work, we also demonstrate that differentiated sphere-derived cell populations not only adopt the immunophenotype of mature spiral ganglio...
Intervention in the molecular interactions that lead to an immune response is possible at various stages of Ag recognition and T cell activation. Perturbation of the interaction of the TCR with the MHC/peptide ligand complex is one... more
Intervention in the molecular interactions that lead to an immune response is possible at various stages of Ag recognition and T cell activation. Perturbation of the interaction of the TCR with the MHC/peptide ligand complex is one approach that has shown promise for autoimmunity and graft rejection in blocking T cell-activated responses. In this study, we investigated the effect of altering the target MHC class I molecule by blocking with Abs. We established a system that analyzed the human T cell response against MHC class I+/class II- porcine stimulatory cell targets. The primary human response against porcine smooth muscle cells was CD8+ T cell dependent. In the presence of F(ab')2 fragments of the MHC class I-reactive Ab, PT-85, the proliferative response was inhibited and production of IL-2 and IFN-gamma was blocked. Moreover, in a secondary response, proliferation was reduced and type 1 cytokine levels were inhibited. In contrast, levels of IL-10 and IL-4 were sustained o...
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Mammalian auditory hair cells do not spontaneously regenerate, unlike hair cells in lower vertebrates, including fish and birds. In mammals, hearing loss due to the loss of hair cells is permanent and intractable. Recent studies in the... more
Mammalian auditory hair cells do not spontaneously regenerate, unlike hair cells in lower vertebrates, including fish and birds. In mammals, hearing loss due to the loss of hair cells is permanent and intractable. Recent studies in the mouse have demonstrated spontaneous hair cell regeneration during a short postnatal period, but this regenerative capacity is lost in the adult cochlea. Reduced regeneration coincides with a transition that results in a decreased pool of progenitor cells in the cochlear sensory epithelium. Here, we review the signaling cascades involved in hair cell formation and morphogenesis of the organ of Corti in developing mammals, the changing status of progenitor cells in the cochlea, and the regeneration of auditory hair cells in adult mammals.
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Hearing loss due to damage to auditory hair cells is normally irreversible because mammalian hair cells do not regenerate. Here, we show that new hair cells can be induced and can cause partial recovery of hearing in ears damaged by noise... more
Hearing loss due to damage to auditory hair cells is normally irreversible because mammalian hair cells do not regenerate. Here, we show that new hair cells can be induced and can cause partial recovery of hearing in ears damaged by noise trauma, when Notch signaling is inhibited by a γ-secretase inhibitor selected for potency in stimulating hair cell differentiation from inner ear stem cells in vitro. Hair cell generation resulted from an increase in the level of bHLH transcription factor Atoh1 in response to inhibition of Notch signaling. In vivo prospective labeling of Sox2-expressing cells with a Cre-lox system unambiguously demonstrated that hair cell generation resulted from transdifferentiation of supporting cells. Manipulating cell fate of cochlear sensory cells in vivo by pharmacological inhibition of Notch signaling is thus a potential therapeutic approach to the treatment of deafness.
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To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. Primary pigmented RPE... more
To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. Primary pigmented RPE harvested from third trimester fetal pigs were transplanted as microaggregates into the subretinal space of 3 albino rabbits. We then used an in situ probe for a repetitive segment of the porcine chromosome to identify the transplanted RPE. Pigmented cells were visible in the subretinal space 2 weeks after transplantation. Approximately 70% of pigment-containing cells were also labeled with the porcine chromosomal marker. Labeled cells were predominantly flatter in morphology and close to Bruch's membrane whereas unlabeled cells were rounder and further from Bruch's membrane. The outer nuclear layer thickness was normal above the pigmented monolayer but was decreased over areas containing multiple layers of pigmented cells. Fetal porcine RPE xenografts can be identified with a nuclear marker for a repetitive segment of the porcine chromosome. The presence of pigment within unlabelled cells suggests that pigment is not a robust marker for transplanted RPE.
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Human CD58 is an adhesion molecule that interacts with CD2 on lymphocytes. We describe here an antibody that blocks responses of human peripheral blood mononuclear cells (PBMCs) to porcine cells and reacts with a porcine protein with... more
Human CD58 is an adhesion molecule that interacts with CD2 on lymphocytes. We describe here an antibody that blocks responses of human peripheral blood mononuclear cells (PBMCs) to porcine cells and reacts with a porcine protein with homology to CD58. Antibodies were isolated with a screen for inhibition of the human antiporcine response. One of these antibodies was used for immunoaffinity purification of a protein that was identified by molecular weight determination, endoglycosidase sensitivity, and microsequencing analysis as a porcine homologue of CD58. The antigen recognized by this antibody was a cell surface protein of relative molecular mass (Mr)=45,000 containing N-linked carbohydrate chains. Immunoaffinity purification of this protein and microsequencing revealed homology to sheep CD58 as well as sequences that were common to this protein and both sheep and human CD58. The protein was widely distributed on porcine cells, including lymphocytes, endothelial cells, muscle cells, and neuronal cells. This antibody efficiently inhibited lysis of porcine targets by human PBMCs in addition to preventing proliferation of the human PBMCs in response to the porcine cells. The CD2 interaction with porcine cells is important for the efficient recognition of porcine tissue, and inhibition of the human antiporcine immune response with the antibody is likely to be caused by the disruption of the human CD2 interaction with this porcine homologue of CD58. The antibody may prove to be useful for the blocking of this interaction without interfering with other functions of T cells.
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Research Interests: Cognitive Science, Stem Cells, Stem Cell, Cell Differentiation, Molecular and cellular biology, and 14 moreCell fusion, Mice, Animals, Bone marrow, Mesenchymal Stem Cell, Inner Ear, Transcription Factor, Biological markers, Cellular and Molecular Neuroscience, Cilia, Cell communication, Neurosciences, Mesenchymal Stromal Cells, and Bone Marrow Cells
Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in... more
Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in vitro after co-culture of de-afferented organ of Corti with spiral ganglion neurons from newborn mice. The glutamatergic synaptic complexes at the ribbon synapse of the inner hair cell contain markers for presynaptic ribbons and postsynaptic densities. We found postsynaptic density protein PSD-95 at the contacts between hair cells and spiral ganglion neurons in newly formed synapses in vitro. The postsynaptic proteins were directly facing the CtBP2-positive presynaptic ribbons of the hair cells. BDNF and NT-3 promoted afferent synaptogenesis in vitro. Direct juxtaposition of the postsynaptic densities with the components of the preexisting ribbon synapse indicated that growing fibers recognized components of the presynaptic sites. Initiation of cochlear synaptogenesis appeared to be influenced by glutamate release from the hair cell ribbons at the presynaptic site since the synaptic regeneration was impaired in glutamate vesicular transporter 3 mutant mice. These insights into cochlear synaptogenesis could be relevant to regenerative approaches for neural loss in the cochlea.
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Research Interests: Skeletal muscle biology, Cell therapy, Animals, Male, Cell Transplantation, and 12 moreClinical Sciences, Rats, Myocardial Infarction, Public health systems and services research, Time Factors, Survival Rate, Circulation, Infarct Size, Heart Ventricles, Left Ventricle, Motor activity, and Cardiac function
Dexamethasone treatment of IM-9 lymphocytes and Fao hepatoma cells resulted in an increase in synthesis of the insulin receptor. The receptors synthesized after stimulation with the glucocorticoid had altered carbohydrate structure. The... more
Dexamethasone treatment of IM-9 lymphocytes and Fao hepatoma cells resulted in an increase in synthesis of the insulin receptor. The receptors synthesized after stimulation with the glucocorticoid had altered carbohydrate structure. The carbohydrate side chains of the insulin receptor were less branched on the dexamethasone-treated cells; i.e., the ratio of saccharides with three and four branches to those bearing only two branches was decreased. The predominant polymannose oligosaccharide after dexamethasone treatment was Man9GlcNAc (vs Man6GlcNAc in the control cell). Both of these changes are consistent with a less complete processing of the N-linked carbohydrate units and were not observed for the total cellular glycoproteins, whereas all glycoproteins manifested an increased sialylation in Fao cells after dexamethasone treatment. These data indicate that glucocorticoid treatment results in alterations in branching of carbohydrate side chains, in the size of polymannose chains and in sialylation of the insulin receptor.
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To characterize the carbohydrate moieties of the insulin receptor on IM-9 lymphocytes, the cells were surface iodinated and solubilized, and the insulin receptors were precipitated with anti-receptor antibody. The precipitates were... more
To characterize the carbohydrate moieties of the insulin receptor on IM-9 lymphocytes, the cells were surface iodinated and solubilized, and the insulin receptors were precipitated with anti-receptor antibody. The precipitates were resuspended, subjected to either enzymatic digestion or chemical treatment with trifluoromethanesulfonic acid and the relative mobilities of the alpha and beta subunits before and after treatment were analyzed by polyacrylamide gel electrophoresis and autoradiography. The results indicate that the alpha subunit possesses primarily N-linked carbohydrate which is both complex (Endoglycosidase F sensitive) and polymannose (Endoglycosidase H sensitive). The beta subunit also contains polymannose oligosaccharide units and has, in addition, a substantial amount of carbohydrate which is removed by chemical treatment but is not susceptible to Endoglycosidase F, suggesting the presence of O-linked saccharides. The apparent molecular weights of the core protein of ...
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Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic... more
Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. W...
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The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr... more
The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr = 20,000) yielded two large glycopeptides (Mr = 9,500 and 7,000) in equimolar amounts which each consisted of one N-linked (Mr = 5,400) and several small O-linked oligosaccharides accounting for a total of nine carbohydrate attachment sites in a 27-amino acid residue segment of the peptide chain. The Pronase treatment of GP-1 (Mr = 100,000) revealed the presence of a large protease-resistant fragment (Mr = 50,000) which contained 34 carbohydrate units (eight N-linked and 26 O-linked) in a segment of 105 amino acids. In addition to these densely glycosylated peptides (one glycosylation site/3 amino acid residues), small glycopeptides with polymannose saccharide units were found in the digests of both proteins. The occurrence of repeating N-acetyllac...
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The heparan sulfate proteoglycan of the bovine glomerular basement membrane (Mr = 200,000, 30% carbohydrate by weight) has been deglycosylated by various chemical and enzymatic procedures to identify the core protein and provide... more
The heparan sulfate proteoglycan of the bovine glomerular basement membrane (Mr = 200,000, 30% carbohydrate by weight) has been deglycosylated by various chemical and enzymatic procedures to identify the core protein and provide information about the N- and O-linked saccharide units. Heparitinase digestion of the proteoglycan reduced its Mr to 143,000, consistent with the removal of its four glycosaminoglycan chains with the exception of short segments adjacent to the carbohydrate-protein linkage region, whereas nitrous acid treatment brought about a smaller reduction in size (to Mr = 168,000) which was shown to be due to the resistance of the internal portion of the heparan sulfate polymer to this reagent. Incubation of the heparitinase-digested proteoglycan with peptide N-glycosidase F decreased its Mr by about 8,000 and liberated oligosaccharides which were primarily acidic in nature; since endo-beta-N-acetylglucosaminidase H did not bring about any saccharide release, it appears...
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Calf thyroid slices were found to incorporate [35S] sulfate into two major plasma membrane glycoproteins, which have been previously designated as GP-1 and GP-3 (Okada, Y., and Spiro, R. G. (1980) J. Biol. Chem. 255, 8865-8872). The... more
Calf thyroid slices were found to incorporate [35S] sulfate into two major plasma membrane glycoproteins, which have been previously designated as GP-1 and GP-3 (Okada, Y., and Spiro, R. G. (1980) J. Biol. Chem. 255, 8865-8872). The 35S-glycoproteins were identified on the basis of their characteristic solubility and electrophoretic migration as well as their affinity for Bandeiraea simplicifolia I lectin. After pronase digestion of these glycoproteins, the 35S-label remained associated with the glycopeptides primarily on asparagine-linked carbohydrate units which were released by hydrazinolysis. Examination of the reduced radio-labeled products obtained by nitrous acid cleavage of the hydrazine-liberated oligosaccharides indicated that sulfate esters of N-acetylglucosamine occurred at three locations on the carbohydrate units; two 35S-monosaccharides (2,5-anhydromannitol 4- and 6-sulfate) and one 35S-disaccharide (beta-Gal(1----4)-2,5-anhydromannitol(6-SO4] were formed. The disacch...
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Liver support using extracorporeal devices and hepatocyte transplantation has received renewed interest for the management of acute and chronic liver failure. The aim of this study was to determine whether xenogeneic porcine hepatocytes... more
Liver support using extracorporeal devices and hepatocyte transplantation has received renewed interest for the management of acute and chronic liver failure. The aim of this study was to determine whether xenogeneic porcine hepatocytes could integrate into the liver parenchyma of cirrhotic Lewis rats when administered by an intrasplenic route. Cirrhosis was induced by carbon tetrachloride (CCl4) inhalation and confirmed histologically. Freshly isolated porcine hepatocytes were infused directly into the splenic pulp at laparotomy over a 5-15-min interval. Using (111)In-labeled hepatocytes, the degree of localization of porcine hepatocytes to the spleen and liver was found to be greater than 60% in both control and cirrhotic rats. Integration of porcine hepatocytes into the rat liver parenchyma was determined by immunohistochemical staining for porcine albumin in rat liver sections. Further confirmation was provided by in situ hybridization using a porcine-specific probe that binds t...
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Elucidation of the mechanism of the immune response against transplanted porcine tissue is critical for the success of xenografting in humans. Both human T cells and NK cells recognize MHC Ags, and human receptors may bind to MHC Ags... more
Elucidation of the mechanism of the immune response against transplanted porcine tissue is critical for the success of xenografting in humans. Both human T cells and NK cells recognize MHC Ags, and human receptors may bind to MHC Ags across species barriers. Molecular characterization of porcine MHC class I clones from two MHC class I loci (P1 and P14) obtained from homozygous inbred miniature swine of three haplotypes (aa, cc, and dd), revealed extensive conservation between loci, suggesting that the genes were products of duplication from a common ancestral sequence. The level of homology between loci was similar to that between the haplotypes at each locus, suggesting that intergenic exchange had limited divergence of these genes. Comparison of the alleles indicated that the polymorphism occurred in the alpha-1 and alpha-2 domains of the class I heavy chain, while the alpha-3 domain was highly conserved among the six genes analyzed. Amino acids in the alpha-2 and alpha-3 domains ...
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Fragmentation of the heparan sulfate chains from bovine glomerular basement membrane (GBM) by hydrazine/nitrous acid treatment followed by NaB3H4-reduction yielded a mixture of six sulfated disaccharides containing D-glucuronic (GlcUA) or... more
Fragmentation of the heparan sulfate chains from bovine glomerular basement membrane (GBM) by hydrazine/nitrous acid treatment followed by NaB3H4-reduction yielded a mixture of six sulfated disaccharides containing D-glucuronic (GlcUA) or L-iduronic acid (IdUA) and terminating in 2,5-anhydro[3H]mannitol (AnManH2), in addition to the nonsulfated component GlcUA beta 1----4AnManH2. Among these products two novel disaccharide units were identified as IdUA alpha 1----4AnManH2(3-SO4) and IdUA(2-SO4)alpha 1----4AnManH2(3-SO4); these accounted for 22% of the total sulfated species indicating that there are 2-3 residues of 3-O-sulfated glucosamine/heparan sulfate chain. The disulfated disaccharide was shown through its release by direct nitrous acid treatment to be situated in a GlcNSO3-IdUA(2-SO4)-GlcNSO3(3-SO4) sequence which is distinct from that in which 3-O-sulfated glucosamine is located in the antithrombin-binding region of heparins. Analyses of heparan sulfate from lens capsule, a n...
Research Interests: Chromatography, Biological Chemistry, Biological Sciences, Basement Membrane, Animals, and 11 moreHigh Pressure Liquid Chromatography, CHEMICAL SCIENCES, Cattle, Glycosaminoglycans, Glucosamine, Lens, Quntitative Thin Layer Chromatography, Disaccharides, Molecular Structure, Carbohydrate Sequence, and Molecular Sequence Data
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ABSTRACT The dehydroalanine containing peptides glycyldehydroalanine and t-butyloxy-carbonyl-L-alanyldehydroalanyl-beta-methyl-D, L-aspartic acid methyl ester were synthesized, respectively, from chloroacetamide and pyruvate and from... more
ABSTRACT The dehydroalanine containing peptides glycyldehydroalanine and t-butyloxy-carbonyl-L-alanyldehydroalanyl-beta-methyl-D, L-aspartic acid methyl ester were synthesized, respectively, from chloroacetamide and pyruvate and from t-butyloxycarbonyl-L-alanyl-S-methyl-L-cysteinyl-beta-methyl-D, L-aspartic acid methyl ester. They were reacted with mercuric acetate in water, 50% methanol and dimethylformamide at 25 degrees and at 80 degrees for various time periods. Reaction proceeded to yield an amino acid amide from the N-terminal side of the unsaturated residue and pyruvate from the C-terminal side. Yields of amino acid amides were 76% for the dipeptide and 40% for the tripeptide. The applicability of the neutral reaction conditions employed to the cleavage of glycoproteins containing both O- and N-glycosidically-linked carbohydrate chains following beta-elimination of the serine and threonine linked oligosaccharides is pointed out.
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In laboratory animals, intrasplenic hepatocyte transplantation corrects the physiologic abnormalities associated with decompensated liver disease. The clinical experience with hepatocyte transplantation for cirrhosis has been... more
In laboratory animals, intrasplenic hepatocyte transplantation corrects the physiologic abnormalities associated with decompensated liver disease. The clinical experience with hepatocyte transplantation for cirrhosis has been disappointing when compared with laboratory experience. The route of hepatocyte delivery may influence hepatocyte engraftment and function. Outbred pigs were recipients of allogeneic pig hepatocytes. Donor hepatocytes were isolated by collagenase perfusion and labeled using 5(6)-carboxyfluorescein diacetate succinimidyl-ester (CMFSE). Cells were introduced into pig spleens by infusion through the splenic artery or by direct splenic puncture. Direct intrasplenic injection produced engraftment that was far superior to that obtained using splenic artery infusion. Splenic artery infusion produced a gastric erosion and large areas of splenic necrosis secondary to vascular occlusion with hepatocytes, whereas direct splenic injection was associated with clinically insignificant intraabdominal hemorrhage. The route of hepatocyte delivery may influence hepatocyte engraftment and explain the disparity in efficacy of hepatocyte transplantation between the laboratory and clinic.
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Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with... more
Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric assay for cell viability, we show that natural antibodies present in the sera of cynomolgus monkey and humans are cytotoxic to porcine endothelial cells in the presence of exogenously added complement. When the terminal alpha-galactosy residues were removed by enzymatic digestion, the cytotoxic effect of natural antibodies on porcine endothelial cells was diminished by > 80%. Evaluation of the time course of reappearance of the alpha-galactosyl epitope at the cell surface revealed that 48 hr after alpha-galactosidase treatment, binding of GS-IB4 was diminished by 60%. These results suggest that glycosidase treatment of cells to be transplanted could prevent hyperacute rejection mediated by natural antibodies.
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In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of... more
In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.