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Biomedical subjects

David M Smith

Publications and source records attributed to David M Smith.

At least 19 recordsLinked to original sources
PubMed2006

In search of radical intermediates in the reactions catalyzed by lysine 2,3-aminomutase and lysine 5,6-aminomutase.

High-level ab initio calculations have been used to study radical intermediates in the reactions catalyzed by lysine 2,3-aminomutase (2,3-LAM) and lysine 5,6-aminomutase (5,6-LAM). The reactions of these enzymes with the substrate analogues 4-oxalysine (4-OL), 4-thialysine (4-TL), or trans-4,5-dehydrolysine (t-4,5-DL) are rationalized in terms of stabilization provided by the substituent to the adjacent radical center. Large changes in the exothermicity accompanying the initial H-abstraction are observed relative to the lysine reference values that follow the series 4-OL < 4-TL < t-4,5-DL. These changes have the primary effect of increasing the endothermicity for subsequent ring-closure to form the putative aziridinylcarbinyl radical intermediate. Such stabilization is consistent with experimental observations of the substrate-derived radical (S*) in the reaction of 2,3-LAM with 4-TL as well as the ability of t-4,5-DL to act as an irreversible inhibitor of 2,3-LAM. Our calculations suggest that 4-TL and trans-3,4-dehydrolysine may also permit experimental characterization of S* radicals in the reactions catalyzed by 5,6-LAM. Strategies for modifying PLP are presented that might lead to the first observation of the aziridinylcarbinyl radical intermediate (I*) in the aminomutase-catalyzed reactions.

Catalysis↗
PubMed2006

Proteasomes and their associated ATPases: a destructive combination.

Protein degradation by 20S proteasomes in vivo requires ATP hydrolysis by associated hexameric AAA ATPase complexes such as PAN in archaea and the homologous ATPases in the eukaryotic 26S proteasome. This review discusses recent insights into their multistep mechanisms and the roles of ATP. We have focused on the PAN complex, which offers many advantages for mechanistic and structural studies over the more complex 26S proteasome. By single-particle EM, PAN resembles a "top-hat" capping the ends of the 20S proteasome and resembles densities in the base of the 19S regulatory complex. The binding of ATP promotes formation of the PAN-20S complex, which induces opening of a gate for substrate entry into the 20S. PAN's C-termini, containing a conserved motif, docks into pockets in the 20S's alpha ring and causes gate opening. Surprisingly, once substrates are unfolded, their translocation into the 20S requires ATP-binding but not hydrolysis and can occur by facilitated diffusion through the ATPase in its ATP-bound form. ATP therefore serves multiple functions in proteolysis and the only step that absolutely requires ATP hydrolysis is the unfolding of globular proteins. The 26S proteasome appears to function by similar mechanisms.

Adenosine Triphosphatases↗
PubMed2006

Learning-related development of context-specific neuronal responses to places and events: the hippocampal role in context processing.

Contextual information plays a key role in learning and memory. Learned information becomes associated with the context such that the context can cue the relevant memories and behaviors. An extensive literature involving experimental brain lesions has implicated the hippocampus in context processing. However, the neurophysiological mechanisms of context coding are not known. Although "context" has typically been defined in terms of the background cues, recent studies indicate that hippocampal neurons are sensitive to subtle changes in task demands, even in an unchanging environment. Thus, the context may also include non-environmental features of a learning situation. In the present study, hippocampal neuronal activity was recorded while rats learned to approach different reward locations in two contexts. Because all of the training took place in the same environment, the contexts were defined by the task demands rather than by environmental stimuli. Learning to differentiate two such contexts was associated with the development of highly context-specific neuronal firing patterns. These included different place fields in pyramidal neurons and different event (e.g., reward) responses in pyramidal and interneurons. The differential firing patterns did not develop in a control condition that did not involve a context manipulation. The context-specific firing patterns could modulate activity in extrahippocampal structures to prime context-appropriate behavioral responses and memories. These results provide direct support for a context processing role of the hippocampus and suggest that the hippocampus contributes contextual representations to episodic memories.

Action Potentials↗
PubMed2006

Insights into the hydrogen-abstraction reactions of diol dehydratase: relevance to the catalytic mechanism and suicide inactivation.

High-level quantum chemistry calculations have been used to examine the hydrogen-abstraction reactions of diol dehydratase (DDH) in the context of both the catalytic mechanism and the enzyme dysfunction phenomenon termed suicide inactivation. The barriers for the catalytic hydrogen-abstraction reactions of ethane-1,2-diol and propane-1,2-diol are examined in isolation, as well as in the presence of various Brønsted acids and bases. Modest changes in the magnitudes of the initial and final abstraction barriers are seen, depending on the strength of the acid or base, and on whether these effects are considered individually or together. The most significant changes (ca. 20 kJ mol(-1)) are found for the initial abstraction barrier when the spectator OH group is partially deprotonated. Kinetic isotope effects including Eckart tunneling corrections (KIEs) have also been calculated for these model systems. We find that contributions from tunneling are of a magnitude similar to that of the contributions from semiclassical theory alone, meaning that quantum effects serve to significantly accelerate the rate of hydrogen transfer. The calculated KIEs for the partially deprotonated system are in qualitative agreement with experimentally determined values. In complementary investigations, the ability of DDH to become deactivated by certain substrate analogues is examined. In all cases, the formation of a stable radical intermediate causes the hydrogen re-abstraction step to become an extremely endothermic process. The consequent inability of 5'-deoxyadenosyl radical to be regenerated breaks the catalytic cycle, resulting in the suicide inactivation of DDH.

Acetaldehyde↗
PubMed2006

Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are related peptides with distinct pharmacological profiles. Calcitonin-receptor-like receptor (CRLR, now known as CL) can function as either an AM receptor or a CGRP receptor, when cotransfected with receptor-activity-modifying proteins (RAMPs) that define ligand-binding specificity. The aim of the present study was to determine the role of endogenously expressed CL (EndoCL) in generating endogenous AM and CGRP receptors. We raised anti-human CL antibody and identified microvascular endothelial cells (MVECs) as a major CL-expressing cell type in tissues by immunohistochemistry. Cultured MVECs continue to express EndoCL as well as fully active endogenous AM- and CGRP-sensitive receptors in vitro, as demonstrated by the ability of both peptides to induce migration and Akt phosphorylation. We therefore tested the hypothesis that endothelial EndoCL can interact with both AM and CGRP by examining receptor internalisation and desensitisation (loss of the ability to induce Akt phosphorylation). We found that agonist-mediated internalisation of EndoCL occurs in response to AM but not CGRP in MVECs. However, AM-induced EndoCL internalisation was blocked by antagonists of both AM and CGRP receptors: AM(22-52) and CGRP(8-37), respectively. Furthermore, AM-induced EndoCL internalisation resulted in desensitisation not only of AM but also of CGRP receptors. Finally, CGRP also induced desensitisation of both endogenous AM and CGRP receptors, but did not mediate EndoCL internalisation despite interaction with this receptor. Thus, EndoCL interacts with both AM and CGRP, and simultaneously acts as a receptor for both peptides (i.e acting as an endogenous AM/CGRP receptor) in endothelial cells. Interaction with either ligand is sufficient to induce EndoCL desensitisation to both AM and CGRP, but differential mechanisms are involved since only AM induces EndoCL internalisation. These novel findings regarding regulation of EndoCL function in endothelial cells are likely to be of importance in conditions where AM or CGRP levels are elevated, such as cardiovascular disease, diabetes and inflammation.

Adrenomedullin↗
PubMed2006

Differential effects of proteasome inhibitors on cell cycle and apoptotic pathways in human YT and Jurkat cells.

Herein, we report differential effects of various proteasome inhibitors including clasto-lactacystin-beta-lactone, (-)-epigallocatechin gallate (EGCG) and N-Acetyl-Leu-Leu-Norleu-al (LLnL) on proteasomal activities of YT and Jurkat cells, human natural killer (NK) and T cell lines, respectively. The inhibitory rates of these inhibitors on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact cells did not show significant differences between the two cell lines. The viability of both cell lines was reduced in the presence of LLnL. Subsequent studies revealed a reduction of the mitochondrial membrane potential and caspase-3 activation in these two cell lines upon treatment with proteasome inhibitors; however, caspase-3 activation occurred much earlier in Jurkat cells. Cell cycle analysis indicated a sub-G(1) apoptotic cell population in Jurkat cells and G(2)/M arrest in YT cells after they were treated by proteasome inhibitors. Moreover, pretreatment of YT cells by a caspase inhibitor followed by a proteasome inhibitor did not increase the percentage of G(2)/M phase cells. In addition, accumulation of p27 and IkappaB-alpha was detected only in Jurkat cells, but not YT cells. In summary, proteasome inhibitors may act differentially in cell cycle arrest and apoptosis of tumors of NK and T cell origin, and may have similar effects on normal NK and T cells.

Apoptosis↗
PubMed2006

Hippocampal place cells, context, and episodic memory.

Although most observers agree that the hippocampus has a critical role in learning and memory, there remains considerable debate about the precise functional contribution of the hippocampus to these processes. Two of the most influential accounts hold that the primary function of the hippocampus is to generate cognitive maps and to mediate episodic memory processes. The well-documented spatial firing patterns (place fields) of hippocampal neurons in rodents, along with the spatial learning impairments observed with hippocampal damage support the cognitive mapping hypothesis. The amnesia for personally experienced events seen in humans with hippocampal damage and the data of animal models, which show severe memory deficits associated with hippocampal lesions, support the episodic memory account. Although an extensive literature supports each of these hypotheses, a specific contribution of place cells to episodic memory has not been clearly demonstrated. Recent data from our laboratory, together with previous findings, indicate that hippocampal place fields and neuronal responses to task-relevant stimuli are highly sensitive to the context, even when the contexts are defined by abstract task demands rather than the spatial geometry of the environment. On the basis of these findings, it is proposed that place fields reflect a more general context processing function of the hippocampus. Hippocampal context representations could serve to differentiate contexts and prime the relevant memories and behaviors. Since episodic memories, by definition, include information about the time and place where the episode occurred, contextual information is a necessary prerequisite for any episodic memory. Thus, place fields contribute importantly to episodic memory as part of the needed context representations. Additionally, recent findings indicate that hippocampal neurons differentiate contexts at progressively finer levels of detail, suggesting a hierarchical coding scheme which, if combined with temporal information, could provide a means of differentiating memory episodes.

Action Potentials↗
PubMed2006

The effects of the ACTIVE cognitive training trial on clinically relevant declines in health-related quality of life.

OBJECTIVES: The Advanced Cognitive Training for Independent and Vital Elderly (ACTIVE) study used three cognitive interventions (memory, reasoning, or speed of processing) in order to improve cognitive abilities. In this article, we evaluate ACTIVE's ability to avoid extensive decline in health-related quality of life (HRQoL). METHODS: ACTIVE enrolled 2,802 adults aged 65 or older and randomized them into one of three cognitive interventions or a no-contact control group. Researchers obtained data on 2,147 participants at the 24-month follow-up. We measured HRQoL by using the eight Short Form-36 scales, and we defined clinically relevant decline on each as a drop of 0.5 standard deviations from baseline. We defined extensive HRQoL decline as clinically relevant drops on four or more Short Form-36 scales, and we assessed this by using multiple logistic regression methods that adjusted for sociodemographic, cognitive, and health status covariates, and incorporated propensity score derived weights in order to adjust for potential attrition bias. RESULTS: We found that 25.0% of ACTIVE participants had extensive HRQoL decline. Participants in the speed-of-processing intervention arm were less likely to have extensive HRQoL decline (adjusted odds ratio = 0.643; p =.004) compared with controls, and participants in the memory and reasoning arms were equivalent to controls (adjusted odds ratios = 1.149 and 1.014, respectively; ps =.322 and.919, respectively). DISCUSSION: Although all three intervention arms improved cognitive ability, only the speed-of-processing arm protected against extensive clinically relevant decline in HRQoL.

Aged↗
PubMed2005

ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins.

The archaeal ATPase complex PAN, the homolog of the eukaryotic 26S proteasome-regulatory ATPases, was shown to associate transiently with the 20S proteasome upon binding of ATP or ATPgammaS, but not ADP. By electron microscopy (EM), PAN appears as a two-ring structure, capping the 20S, and resembles two densities in the 19S complex. The N termini of the archaeal 20S alpha subunits were found to function as a gate that prevents entry of seven-residue peptides but allows entry of tetrapeptides. Upon association with the 20S particle, PAN stimulates gate opening. Although degradation of globular proteins requires ATP hydrolysis, the PAN-20S complex with ATPgammaS translocates and degrades unfolded and denatured proteins. Rabbit 26S proteasomes also degrade these unfolded proteins upon ATP binding, without hydrolysis. Thus, although unfolding requires energy from ATP hydrolysis, ATP binding alone supports ATPase-20S association, gate opening, and translocation of unfolded substrates into the proteasome, which can occur by facilitated diffusion through the ATPase.

Adenosine Triphosphatases↗
PubMed2005

Ras mutation impairs epithelial barrier function to a wide range of nonelectrolytes.

Although ras mutations have been shown to affect epithelial architecture and polarity, their role in altering tight junctions remains unclear. Transfection of a valine-12 mutated ras construct into LLC-PK1 renal epithelia produces leakiness of tight junctions to certain types of solutes. Transepithelial permeability of D-mannitol increases sixfold but transepithelial electrical resistance increases >40%. This indicates decreased paracellular permeability to NaCl but increased permeability to nonelectrolytes. Permeability increases to D-mannitol (Mr 182), polyethylene glycol (Mr 4000), and 10,000-Mr methylated dextran but not to 2,000,000-Mr methylated dextran. This implies a "ceiling" on the size of solutes that can cross a ras-mutated epithelial barrier and therefore that the increased permeability is not due to loss of cells or junctions. Although the abundance of claudin-2 declined to undetectable levels in the ras-overexpressing cells compared with vector controls, levels of occludin and claudins 1, 4, and 7 increased. The abundance of claudins-3 and -5 remained unchanged. An increase in extracellular signal-regulated kinase-2 phosphorylation suggests that the downstream effects on the tight junction may be due to changes in the mitogen-activated protein kinase signaling pathway. These selective changes in permeability may influence tumorigenesis by the types of solutes now able to cross the epithelial barrier.

Animals↗
PubMed2005

Divergent mechanisms of suicide inactivation for ethanolamine ammonia-lyase.

Ab initio molecular orbital calculations have been used to study the mechanism of suicide inactivation of ethanolamine ammonia-lyase induced by three different substrate analogues. Analysis of the normal catalytic mechanism with 2-aminoethanol (ethanolamine) as substrate predicts that both the hydrogen-abstraction and hydrogen-reabstraction steps involving the B(12)-cofactor are likely to be exothermic. On the other hand, the proposed inactivation mechanism for the first substrate analogue, glycolaldehyde, leads to a highly stabilized radical that results in a very endothermic (by ca. 90 kJ mol(-)(1)) hydrogen-reabstraction step, which is thought to halt the normal function of the enzyme. Curiously, the energy requirements for a catalytically imposed mechanism in the case of the second substrate analogue, 2-hydroxyethylhydrazine (HEH), parallel those for the catalytic substrate, despite the fact that HEH is found to inactivate EAL experimentally. However, further analysis reveals the presence of a lower energy pathway for HEH that leads to the formation of the highly stabilized hydrazinium radical cation. In a manner similar to when glycolaldehyde is the substrate analogue, this results in an endothermicity for the hydrogen-reabstraction step that is prohibitively large. In contrast to these related inactivation mechanisms, the third substrate analogue, 2-aminoacetaldehyde, apparently accomplishes the inactivation of EAL in an entirely different manner. A pathway for the experimentally observed formation of acetic acid and ammonium cation has been identified and appears catalytic in the sense that 5'-deoxyadenosyl radical is regenerated. However, mechanisms to account for the subsequent formation of 4',5'-anhydroadenosine and degradation of the corrinoid ring of the cofactor have not been elucidated.

Acetaldehyde↗
PubMed2005

Terrestrial gamma-ray flashes observed up to 20 MeV.

Terrestrial gamma-ray flashes (TGFs) from Earth's upper atmosphere have been detected with the Reuven Ramaty High Energy Solar Spectroscopic Imager (RHESSI) satellite. The gamma-ray spectra typically extend up to 10 to 20 megaelectron volts (MeV); a simple bremsstrahlung model suggests that most of the electrons that produce the gamma rays have energies on the order of 20 to 40 MeV. RHESSI detects 10 to 20 TGFs per month, corresponding to approximately 50 per day globally, perhaps many more if they are beamed. Both the frequency of occurrence and maximum photon energy are more than an order of magnitude higher than previously known for these events.

Journal Article↗
PubMed2005

Vibration-rotation interactions between overtone and combination levels of asymmetric-top molecules: application to the infrared spectroscopy of formaldehyde and ketene.

The conventional vibration-rotation Hamiltonian for an asymmetric-top molecule is rewritten by expanding the elements of the inverse inertial tensor about the equilibrium molecular geometry. The approach allows the identification of terms in the Hamiltonian that couple states differing by two, three, or four vibrational quanta and hence the calculation of dimensioned Coriolis xi coupling coefficients for interacting fundamental, overtone, and combination levels. The matrix elements that result from the application of the expanded Hamiltonian depend upon the harmonic vibrational wave numbers, equilibrium moments of inertia, Coriolis zeta parameters, and the derivatives of the elements of the inertial tensor matrix with respect to each of the normal coordinates. The Coriolis coupling coefficients may be calculated through evaluation of the summations that result from the appropriate terms. The validity of the approach is demonstrated through the calculation of coupling coefficients for interacting levels in formaldehyde and ketene. The uncertainty in the calculated values of the coupling coefficients is typically better than +/-6%, although the values calculated for interactions that involve low-frequency vibrational modes are less reliable. Comparisons are made between the calculated values and experimental results.

Journal Article↗
PubMed2005

Naturally occurring proteasome inhibitors from mate tea (Ilex paraguayensis) serve as models for topical proteasome inhibitors.

Proteasome inhibitors have emerged as a clinically important therapy for neoplastic disease, with velcade, an organoboron compound used extensively in multiple myeloma. Recently, (-)-epigallocatechin gallate has been found to be a potent inhibitor of the proteasomal chymotrypsin-like activity. Other compounds that inhibit angiogenesis and are active as chemopreventive agents, such as curcumin, also inhibit proteasome activity. We have screened natural product extracts using ras-transformed endothelial cells (SVR cells) as a bioassay, and found that extracts of mate tea (Ilex paraguayensis) inhibit the growth of these endothelial cells. The extract was fractionated and found to have novel cinnamate esters that inhibit proteasome activity. Based upon the structures of the compounds isolated from mate tea, we examined synthetic analogs of these compounds for proteasome activity. Cinnamic acid amides had no inhibitory activity against proteasomes, whereas cinnamate esters displayed the activity. Based upon these findings, preclinical and clinical trials of topical cinnamate esters as proteasome inhibitors are warranted for psoriasis and other inflammatory disorders.

Cell Division↗
PubMed2005

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors.

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241, Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide, N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P, predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Amino Acid Sequence↗
PubMed2004

Suicide inactivation of dioldehydratase by glycolaldehyde and chloroacetaldehyde: an examination of the reaction mechanism.

High-level ab initio calculations have been used to study the mechanism for the inactivation of diol dehydratase (DDH) by glycolaldehyde or 2-chloroacetaldehyde. As in the case of the catalytic substrates of DDH, e.g., ethane-1,2-diol, the 5'-deoxyadenosyl radical (Ado*) is able to abstract a hydrogen atom from both substrate analogues in the initial step on the reaction pathway, as evidenced by comparable energy barriers. However, in subsequent step(s), each substrate analogue produces the highly stable glycolaldehyde radical. The barrier for hydrogen atom reabstraction by the glycolaldehyde radical is calculated to be too high ( approximately 110 kJ mol-1) to allow Ado* to be regenerated and recombine with the cob(II)alamin radical, the latter therefore remaining tightly bound to DDH. As a consequence, the catalytic pathway is disrupted, and DDH becomes an impotent enzyme. Interconversion of equivalent structures of the glycolaldehyde radical via the symmetrical cis-ethanesemidione radical is calculated to require 38 kJ mol-1. EPR indications of a symmetrical cis-ethanesemidione structure are likely to be the result of formation of an equilibrium mixture of glycolaldehyde radical structures, this equilibration being facilitated by partial deprotonation of the glycolaldehyde radical by the carboxylate of an amino acid residue within the active site of DDH.

Acetaldehyde↗
PubMed2004

The unusual bifunctional catalysis of epimerization and desaturation by carbapenem synthase.

High-level ab initio calculations have been used to study the mechanism for the conversion of (3S,5S)-carbapenam to the biologically active beta-lactam antibiotic, (5R)-carbapenem, catalyzed by carbapenem synthase. This process involves epimerization at C5 and desaturation at C2/C3. Our calculations suggest that the reaction proceeds via initial abstraction of the C5 hydrogen atom, followed by epimerization. In addition, we have identified an attractive mechanism for coupling the epimerization and desaturation in thermodynamically favorable steps with the aid of an external reductant. Other mechanisms that have been examined have significantly higher energy requirements or do not appear to be consistent with available experimental evidence.

Carbapenems↗