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(1 - 7 of 7)
- Title
- Structure-function relationships in eukaryotic and prokaryotic family 6 glycosyltransferases.
- Creator
- Tumbale, Percy., Charles E. Schmidt College of Medicine, Department of Biomedical Science
- Abstract/Description
-
Carbohydrate Active Enzyme family 6 (CA6) glycosyltransferases (GTs) are type II transmembrane proteins localized in the Golgi apparatus. CA6 GTs have a GT-A fold, a type of structure that resembles the Rossman fold and catalyze the transfer either galactose (Gal) or N-acetylgalactosamine (GalNAc) from the UDP nucleotide sugar to an non-reducing terminal Gal or GalNAc on an acceptor via an a-1,3 linkage. In this reaction, the anomeric configuration of the sugar moiety of the donor is retained...
Show moreCarbohydrate Active Enzyme family 6 (CA6) glycosyltransferases (GTs) are type II transmembrane proteins localized in the Golgi apparatus. CA6 GTs have a GT-A fold, a type of structure that resembles the Rossman fold and catalyze the transfer either galactose (Gal) or N-acetylgalactosamine (GalNAc) from the UDP nucleotide sugar to an non-reducing terminal Gal or GalNAc on an acceptor via an a-1,3 linkage. In this reaction, the anomeric configuration of the sugar moiety of the donor is retained in the product. CA6 GTs includes the histo-blood group A and B GTs, a-galactosyltransferase (a3GT), Forssman glycolipid synthase (FS), isogloboside 3 synthase (iGb3) in mammals. a3GT and its products (a-Gal epitode) are present in most mammals but are absent in humans and old world primates because of inactivating mutations. The absence of a3GT and its products results in the production of anti-a-Gal epitope natural antibodies in these species., Up to date, the catalytic mechanisms of the CA6 GTs are not well understood. Based on previous structural and mutagenesis studies of bovine aB3GT, we investigated active site residues (His315, Asp316, Ser318, His319, and Lys359) that are highly conserved among CA6 GTs. We have also investigated the role of the C-terminal region by progressive C-terminal truncations. Findings from these studies clarify the functional roles of these residues in structure, catalysis, and specificity in these enzymes and have implications for their catalytic mechanisms. GTs are useful tools in synthesis of glycans for various applications in science and medicine. Methods for the large scale production of pure glycans are continuously being developed. We created a limited randomized combinatorial library based on knowledge of structural information and sequence analysis of the enzyme and its mammalian homologues., Two GalNAc-specific variants were identified from the library and one Glc-specific variant was identified by site-direct mutagenesis. The glycosyltransferase activities of these variants are expected to be improved by further screens of libraries which are designed using the variants as templates. The mammalian CA6 GTs that have been characterized to date are metal-independent and require the divalent cation, Mn2+ for activity. In some recently-discovered bacterial CA6 GTs, the DXD sequence that is present in eukaryotic GTs is replaced by NXN. We cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that has been linked with inflammatory bowel disease. Functional characterization shows it is a metal-independent monomeric GT that efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A glycan., Mutational studies indicated that despite the lack of a metal cofactor there are similarities in structure-function relationships between the bacterial and vertebrate family 6 GTs.
Show less - Date Issued
- 2009
- PURL
- http://purl.flvc.org/FAU/186686
- Subject Headings
- Molecular biology, Mathematical models, Glycotransferase genes, Biological transport, Proteins, Synthesis, Evolutionary genetics
- Format
- Document (PDF)
- Title
- An investigation of membrane transporter proteins in the distal vertebrate retina: excitatory amino acid transporters and sodium potassium chloride cotransporters.
- Creator
- Purpura, Lauren Angeline, Shen, Wen, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biological Sciences
- Abstract/Description
-
Neurons are able to maintain membrane potential and synaptic integrity by an intricate equilibrium of membrane transporter proteins and ion channels. Two membrane proteins of particular importance in the vertebrate retina are the excitatory amino acid transporters (EAATs) which are responsible for the reuptake of glutamate into both glial and neuronal cells and the sodium potassium chloride cotransporters (NKCCs) that are responsible for the uptake of chloride ions into the cell. NKCCs are...
Show moreNeurons are able to maintain membrane potential and synaptic integrity by an intricate equilibrium of membrane transporter proteins and ion channels. Two membrane proteins of particular importance in the vertebrate retina are the excitatory amino acid transporters (EAATs) which are responsible for the reuptake of glutamate into both glial and neuronal cells and the sodium potassium chloride cotransporters (NKCCs) that are responsible for the uptake of chloride ions into the cell. NKCCs are electro-neutral with the uptake of 2 Cl- coupled to an exchange of a potassium and Na+ ion into the cells. Therefore, there is little change of cell membrane potential in the action of NKCCs. In this study the localization and function of EAATs in the distal retina is investigated. Whole cell patch clamp recordings in lower vertebrate retina have demonstrated that EAAT2 is the main synaptic EAATs in rod photoreceptors and it is localized to the axon terminals. Furthermore, the action of the transporter seems to be modified by intracellular calcium concentration. There is also evidence that EAAT2 might be regulated by feedback from the neuron network by glycinergic and GABAergic mechanisms. The second half of this study investigates expression of NKCCs in the retina by western blot analysis and quantitative polymerase chain reaction. There are two forms of NKCCs, NKCC1 and NKCC2. NKCC1 is mostly expressed in the central nervous system and NKCC2 was thought to only be expressed in the kidneys. NKCC1 is responsible for the majority of chloride uptake into neuronal and epithelial cells and NKCC1 is expressed in the distal retina where photoreceptors synapse on second order horizontal and bipolar cells. This study found the expression of NKCC1 in the distal retina to be regulated by temporal light and dark adaptation. Light adaptation increased phosphorylated NKCC1 expression (the active form of the cotransporter). The increase in NKCC1 expression during light adaptation was modulated by dopamine. Specifically, a D1 receptor agonist increased phosphorylated NKCC1 expression. Dopamine is an essential chemical and receptor known for initiating light adaptation in retina. Finally, an NKCC1 knockout mouse model was examined and it revealed that both forms of NKCC are expressed in the vertebrate retina.
Show less - Date Issued
- 2014
- PURL
- http://purl.flvc.org/fau/fd/FA00004224, http://purl.flvc.org/fau/fd/FA00004224
- Subject Headings
- Biological transport, Carrier proteins, Cellular signal transduction, Neural receptors, Retina -- Cytology
- Format
- Document (PDF)
- Title
- Influence of small conductance calcium-activated potassium channels (SK,Kca2) on long-term memory: global and local analysis across time- and task- dependent measures.
- Creator
- Vick, Kyle A., Charles E. Schmidt College of Science, Department of Psychology
- Abstract/Description
-
Small conductance calcium-activated potassium (SK) channels are found ubiquitously throughout the brain and modulate the encoding of learning and memory. Systemic injection of 1-ethyl-2-benzimidalzolinoe (EBIO), a SK channel activator, impairs the encoding of novel object memory and locomotion but spares fear memory encoding in C57BL/6NHsd mice. The memory impairments discovered were not due to non-cognitive performance confounds such as ataxia, anxiety, attention or analgesia. Further...
Show moreSmall conductance calcium-activated potassium (SK) channels are found ubiquitously throughout the brain and modulate the encoding of learning and memory. Systemic injection of 1-ethyl-2-benzimidalzolinoe (EBIO), a SK channel activator, impairs the encoding of novel object memory and locomotion but spares fear memory encoding in C57BL/6NHsd mice. The memory impairments discovered were not due to non-cognitive performance confounds such as ataxia, anxiety, attention or analgesia. Further investigation with intra-hippocampal application of EBIO revealed SK channels in dorsal CA1 contribute to the encoding deficits seen systemically, but do not account for the full extent of the impairment. Concentrated activation of dorsal CA1 SK channels do not influence fear memory encoding or locomotor impairments. Taken together, these data indicate SK channels, especially in the dorsal hippocampus, have a modulatory role on novel object memory encoding, but not retrieval; however, pharmacological activation of hippocampal SK channels does not appear to influence fear memory encoding.
Show less - Date Issued
- 2009
- PURL
- http://purl.flvc.org/FAU/192991
- Subject Headings
- Mice as laboratory animals, Cellular signal transduction, Memory, Research, Biological transport, Research, Potassium channels, Physiological effect
- Format
- Document (PDF)
- Title
- New insights into the neuromodulatory role and potential action site of taurine in retinal neurons.
- Creator
- Bulley, Simon, Charles E. Schmidt College of Science, Department of Biomedical Science
- Abstract/Description
-
Taurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study...
Show moreTaurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study the neuromodulatory role of taurine in the CNS and to gain insights into its potential sites of action. A combination of techniques was used, including whole-cell patch clamp recording to study taurine's regulation of voltage-gated potassium (K+) and Ca2+ channels and Fluo-4AM Ca2+-imaging to study taurine's regulation of glutamate-induced [Ca2+] I,. Taurine was shown to suppress of glutamate-induced [Ca2+] l, in a dose dependent manner. This suppression was mostly sensitive to the glycine rece ptor antagonist Strychnine but insensitive to any GABA receptor antagonist. The remaining strychnine-insensitive effect was inhibited with the protein kinase A (PKA) inhibitor, PKI, suggesting that there was an additional metabotropic pathway. Moreover, using the protein kinase C (PKC) inhibitor, GF109203X, there was an enhancement in strychnine-insensitive taurine's regulation. Taurine inhibits voltage-gated Ca2+ channels in the retinal neurons and has a dual effect on voltage-gated K+ channels. Taurine causes an increase in K+ current amplitude which is further enhanced with PKI and blocked with GF109203X, suggesting that it is through a PKC-dependent pathway negatively controlled by PKA-dependent pathway., There is a suppression of K+ current by taurine with intracellular application of GF109203X, suggesting that the reduction is through a PKA-dependent pathway. With both PKC and PKA inhibitors there is no longer an enhancement in maximum amplitude but a shift of volt dependence on a hyperpolarizing direction. Taurine's enhancement of K+ current is blocked by the Kv1.3 subtype antagonist Margatoxin, with Kv1.3 accounting for the majority of delayed-rectifier sustained current in bipolar and amacrine cells, as well as 50% of ganglion cells. Interestingly, the enhancement of K+ current by taurine is blocked by 5HT2A antagonist MDL11939, suggesting that activation of PKC is through this metabotropic serotonin receptor subtype. The suppression of voltage-gated Ca2+ channels is reversed with a combination of MDL11939 and the 5HT1A antagonist NAN-190. These results provide the evidence that the natural effect of taurine in the retinal neurons might be dependent on the activation of both 5HT1A and 5HT2A receptors. The high apparent activity of taurine on 5HT receptors could have important implication for the actions of taurine in central brain in which taurine has been known to be beneficial for improving mental health, as well as learning and memory processes.
Show less - Date Issued
- 2010
- PURL
- http://purl.flvc.org/FAU/2953206
- Subject Headings
- Biological transport, Eye, Physiology, Taurine, Physiological effect, Taurine, Therapeutic use, Central nervous system, Physiology
- Format
- Document (PDF)
- Title
- The effect of small conductance calcium-activated potassium channels on emotional learning and memory.
- Creator
- Sanguinetti, Shannon, Stackman, Robert W., Florida Atlantic University, Charles E. Schmidt College of Science, Department of Psychology
- Abstract/Description
-
Small conductance Ca2+-activated K+ (SK) channels have been shown to alter the encoding of spatial and non-spatial memory in the hippocampus by shaping glutamatergic postsynaptic potentials and modulating NMDA receptor-dependent synaptic plasticity. When activated, dendritic SK channels reduce hippocampal neuronal excitability and LTP. Similar SK channel properties have been demonstrated in lateral amygdala (LA) pyramidal neurons. Additionally, induction of synaptic plasticity and beta...
Show moreSmall conductance Ca2+-activated K+ (SK) channels have been shown to alter the encoding of spatial and non-spatial memory in the hippocampus by shaping glutamatergic postsynaptic potentials and modulating NMDA receptor-dependent synaptic plasticity. When activated, dendritic SK channels reduce hippocampal neuronal excitability and LTP. Similar SK channel properties have been demonstrated in lateral amygdala (LA) pyramidal neurons. Additionally, induction of synaptic plasticity and beta-adrenoreceptor activation in LA pyramidal neurons causes PKA-mediated internalization of SK channels from the postsynaptic density. Chronic activation of the amygdala through repetitive stressful stimuli can lead to excitatory synaptic strengthening that may create permanent hyper-excitability in its circuitry. This mechanism may contribute to a number of mood and anxiety disorders. The selective influence of SK channels in the LA on anxiety and fear conditioning are not known. The thesis project outlined herein examined whether SK channel blockade by bee venom peptide, apamin, during a repetitive acute fear conditioning paradigm was sufficient to alter fear memory encoding and the resulting behavioral outcome. Following the final fear memory test session, mice were tested in the open field immediately after the second fear conditioning test session. The findings indicate that intracranial LA microinfusions of apamin did not affect memory encoding or subsequent anxiety.
Show less - Date Issued
- 2015
- PURL
- http://purl.flvc.org/fau/fd/FA00004543, http://purl.flvc.org/fau/fd/FA00004543
- Subject Headings
- Biological transport -- Research, Cellular signal transduction, Memory -- Research, Mice as laboratory animals, Potassium channels -- Physiological effect
- Format
- Document (PDF)
- Title
- Assay development for lysyl hydroxylase.
- Creator
- Patel, Deepak A., Florida Atlantic University, Fields, Gregg B.
- Abstract/Description
-
Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the...
Show moreHydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine-containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.
Show less - Date Issued
- 2006
- PURL
- http://purl.flvc.org/fcla/dt/13384
- Subject Headings
- Biological transport, Proteins--Metabolism, Peptides--Analysis, Coenzymes, Bioorganic chemistry
- Format
- Document (PDF)
- Title
- The Comparison of High-Intensity Interval Exercise vs. Continuous Moderate Exercise on C1q/TNF-Related Protein-9 Expression and Flow-Mediated Vasodilation.
- Creator
- Fico, Brandon G., Huang, Chun-Jung, Florida Atlantic University, College of Education, Department of Exercise Science and Health Promotion
- Abstract/Description
-
The primary purpose of this study was to investigate the effect of acute high-intensity interval exercise (HIIE) vs. continuous moderate-intensity exercise (CME) on serum CTRP9 and brachial FMD responses in obese and normal-weight subjects. Sixteen participants (9 obese and 7 normal-weight) completed HIIE and CME in a randomized fashion. Our results showed a significant time effect for CTRP9 immediately following acute HIIE and CME in both groups. Furthermore, both significant treatment by...
Show moreThe primary purpose of this study was to investigate the effect of acute high-intensity interval exercise (HIIE) vs. continuous moderate-intensity exercise (CME) on serum CTRP9 and brachial FMD responses in obese and normal-weight subjects. Sixteen participants (9 obese and 7 normal-weight) completed HIIE and CME in a randomized fashion. Our results showed a significant time effect for CTRP9 immediately following acute HIIE and CME in both groups. Furthermore, both significant treatment by time and group by time interactions for FMD were observed following both exercise protocols, with greater CME-induced FMD response in obese subjects than normal-weight subjects. Additionally, a positive correlation in percent change (baseline to peak) between CTRP9 and FMD was observed following acute CME. These findings support acute CME for improvement of endothelial function in obesity. Furthermore, the novel results from this study provide a foundation for additional examination of the mechanisms of exercise-mediated CTRP9 on endothelial function.
Show less - Date Issued
- 2017
- PURL
- http://purl.flvc.org/fau/fd/FA00004915, http://purl.flvc.org/fau/fd/FA00004915
- Subject Headings
- Cardiovascular system--Physiology., Biological transport., Exercise--Physiological aspects., Interval training--Physiological aspects., Bioinformatics., Exercise--Health aspects., Lifestyles--Health aspects., Gene expression.
- Format
- Document (PDF)
