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Biomedical subjects

Steven Smith

Publications and source records attributed to Steven Smith.

15 recordsLinked to original sources
PubMed2006

RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists.

Prostacyclin (PGI2) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794).RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pKi) were 9.3 +/- 0.1 and 7.7 +/- 0.03, respectively; in a recombinant IP receptor system, pKi values were 8.7 +/- 0.06 and 6.9 +/- 0.1, respectively. Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinities (pKi) of RO1138452 and RO3244794 were 9.0 +/- 0.06 and 8.5 +/- 0.11, respectively. Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pKi values for EP1 (< 5), EP3 (5.38), EP4 (5.74) and TP (5.09). RO1138452 (1-10 mg kg(-1), i.v.) and RO3244794 (1-30 mg kg(-1), i.v.) significantly reduced acetic acid-induced abdominal constrictions. RO1138452 (3-100 mg kg(-1), p.o.) and RO3244794 (0.3-30 mg kg(-1), p.o.) significantly reduced carrageenan-induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg(-1), p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti-inflammatory potential.

Animals↗
PubMed2006

Investigating interpersonal competencies of cardiac surgery teams.

Successful cardiac surgery requires highly skilled individuals to interact effectively in a variety of complex situations. Although cardiac surgery requires individuals to have the requisite medical knowledge and skills, interpersonal competencies are vital to any successful cardiac surgery. Surgeons, anesthesiologists, perfusionists, nurses and residents must communicate effectively in order to ensure a successful patient outcome. Breakdowns in communication, decision-making or leadership could lead to adverse patient outcomes. Realizing that human error is responsible for many adverse patient outcomes, we attempted to understand the team processes involved in cardiac surgery. An adaptation of the Operating Room Management Attitudes Questionnaire was used to gather a variety of responses related to group decision-making and communication. The results indicate inherent group differences based on factors such as seniority and occupational group membership. The implications of the research findings and suggestions for future research are discussed in detail.

Attitude of Health Personnel↗
PubMed2005

Comparison of the predicted structures of loops in the ras-SOS protein bound to a single ras-p21 protein with the crystallographically determined structures in SOS bound to two ras-p21 proteins.

We have previously computed the structures of three loops, residues 591-596, 654-675 and 742-751, in the ras-p21 protein-binding domain (residues 568-1044) of the guanine nucleotide-exchange-promoting SOS protein that were crystallographically undefined when one molecule of ras-p21 (unbound to nucleotide) binds to SOS. Based on our computational results, we synthesized three peptides corresponding to sequences of each of these three loops and found that all three peptides strongly inhibit ras-p21 signaling. More recently, a new crystal structure of SOS has been determined in which this protein binds to two molecules of ras-p21, one unbound to GTP and one bound to GTP. In this structure, the 654-675 loop and residues 742-743 and 750-751 are now crystallographically defined. We have superimposed our energy-minimized structure of the ras-binding domain of SOS bound to one molecule of ras-p21 on the X-ray structure for SOS bound to two molecules of ras-p21. We find that, while the two structures are superimposable, there are large deviations of the residues 673 and 676 and 741 and 752, flanking the two loop segments. This suggests that the binding of the extra ras-p21 molecule, which is far from each of the three loops, induces conformational changes in these domains and further supports their role in signal transduction. In spite of these differences, we have superimposed our computed structures for the loop residues on those from the more recent X-ray structure. Our structure for the 654-675 segment is an anti-parallel beta-sheet with a reverse turn at residues 663-665; in the X-ray structure residues 655-662 adopt an alpha-helical conformation; on the other hand, our computed structure for residues 663-675 superimpose on the X-ray structure for these residues. We further find that our computed structures for residues 742-743 and 750-751 are superimposable on the X-ray structure for these residues.

Animals↗
PubMed2005

Distribution of the vitamin D receptor and 1 alpha-hydroxylase in human brain.

Despite a growing body of evidence that Vitamin D is involved in mammalian brain functioning, there has been a lack of direct evidence about its role in the human brain. This paper reports, for the first time, the distribution of the 1,25-dihydroxyvitamin D3 receptor (VDR), and 1alpha-hydroxylase (1alpha-OHase), the enzyme responsible for the formation of the active vitamin in the human brain. The receptor and the enzyme were found in both neurons and glial cells in a regional and layer-specific pattern. The VDR was restricted to the nucleus whilst 1alpha-OHase was distributed throughout the cytoplasm. The distribution of the VDR in human brain was strikingly similar to that reported in rodents. Many regions contained equivalent amounts of both the VDR and 1alpha-OHase, however the macrocellular cells within the nucleus basalis of Meynert (NBM) and the Purkinje cells in the cerebellum expressed 1alpha-OHase in the absence of VDR. The strongest immunohistochemical staining for both the receptor and enzyme was in the hypothalamus and in the large (presumably dopaminergic) neurons within the substantia nigra. The observed distribution of the VDR is consistent with the proposal that Vitamin D operates in a similar fashion to the known neurosteroids. The widespread distribution of 1alpha-OHase and the VDR suggests that Vitamin D may have autocrine/paracrine properties in the human brain.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2005

Reduction of astrogliosis by early treatment of pneumococcal meningitis measured by simultaneous imaging, in vivo, of the pathogen and host response.

We developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and the accompanying neuronal injury in live transgenic mice. Streptococcus pneumoniae engineered for bioluminescence (lux) was used for direct visualization of disease progression and antibiotic treatment in a mouse model of meningitis. The host response was monitored in transgenic mice containing an inducible firefly luciferase (luc) reporter gene under transcriptional control of the mouse glial fibrillary acidic protein (GFAP) promoter. Based on the different spectra of light emission and substrate requirements for lux and luc, we were able to separately monitor the two reporters using a highly sensitive in vivo imaging system. The level of neuronal damage and recovery following antibiotic treatment was dependent on the time of treatment. This model has potential for simultaneous multiparameter monitoring and testing of therapies that target the pathogen or host response to prevent neuronal injury and recovery.

Animals↗
PubMed2004

A self-tuning method for one-chip SNP identification.

Current methods for interpreting oligonucleotide-based SNP-detection microarrays, SNP chips, are based on statistics and require extensive parameter tuning as well as extremely high-resolution images of the chip being processed. We present a method, based on a simple data-classification technique called nearest-neighbors that, on haploid organisms, produces results comparable to the published results of the leading statistical methods and requires very little in the way of parameter tuning. Furthermore, it can interpret SNP chips using lower-resolution scanners of the type more typically used in current microarray experiments. Along with our algorithm, we present the results of a SNP-detection experiment where, when independently applying this algorithm to six identical SARS SNP chips, we correctly identify all 24 SNPs in a particular strain of the SARS virus, with between 6 and 13 false positives across the six experiments.

Algorithms↗
PubMed2004

Single nucleotide polymorphism (SNP) discovery in mammals: a targeted-gene approach.

Single nucleotide polymorphisms (SNPs) have rarely been exploited in nonhuman and nonmodel organism genetic studies. This is due partly to difficulties in finding SNPs in species where little DNA sequence data exist, as well as to a lack of robust and inexpensive genotyping methods. We have explored one SNP discovery method for molecular ecology, evolution, and conservation studies to evaluate the method and its limitations for population genetics in mammals. We made use of 'CATS' (or 'EPIC') primers to screen for novel SNPs in mammals. Most of these primer sets were designed from primates and/or rodents, for amplifying intron regions from conserved genes. We have screened 202 loci in 16 representatives of the major mammalian clades. Polymerase chain reaction (PCR) success correlated with phylogenetic distance from the human and mouse sequences used to design most primers; for example, specific PCR products from primates and the mouse amplified the most consistently and the marsupial and armadillo amplifications were least successful. Approximately 24% (opossum) to 65% (chimpanzee) of primers produced usable PCR product(s) in the mammals tested. Products produced generally high but variable levels of readable sequence and similarity to the expected genes. In a preliminary screen of chimpanzee DNA, 12 SNPs were identified from six (of 11) sequenced regions, yielding a SNP on average every 400 base pairs (bp). Given the progress in genome sequencing, and the large numbers of CATS-like primers published to date, this approach may yield sufficient SNPs per species for population and conservation genetic studies in nonmodel mammals and other organisms.

Animals↗
PubMed2003

The functional role of the JAK-STAT pathway in post-infarction remodeling.

OBJECTIVES: Recently, the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway was found to be prominently associated with activation of the autocrine loop of the heart tissue-localized renin angiotensin system (RAS). We investigated if the JAK-STAT pathway is activated in the post-myocardial infarction (MI) non-ischemic myocardium (NIM), destined to undergo remodeling and whether blockade of the pathway in vivo can modify early post-MI remodeling. METHODS: We investigated the time course of tyrosine phosphorylation of JAK-STAT and gp130 proteins in the NIM of post-MI rat heart as well as the binding activity of STAT proteins to the St-domain of the angiotensinogen gene promoter. We further compared the effects of in vivo blockade of RAS by the AT(1) receptor (AT(1)R) blocker losartan with the in vivo blockade of JAK-STAT pathway by the specific JAK2 blocker tyrphostin AG490 on certain aspects of early post-MI remodeling. RESULTS: We showed that JAK2, STATs 1, 3, 5a and 6 and gp130 proteins are tyrosine phosphorylated as early as 5-30 min post-MI and that STATs 1, 3, and 5a remain activated up to 7 days post-MI. Gel mobility shift assay showed a strong binding activity of STAT proteins to the St-domain of angiotensinogen gene promoter in 1-day post-MI NIM. The binding was significantly reduced in rat hearts previously treated with losartan or tyrphostin AG490. Supershift experiments identified STATs 3 and 5a as specifically interacting with the St-domain. Both AT(1)R and JAK2 blockade resulted in significant amelioration of the increase of protein phosphatase 1 activity and decrease in basal level of p16-phospholamban that may underlie early diastolic dysfunction, as well as partial amelioration of early downregulation of Kv4.2 gene expression that may underlie increased arrhythmogenicity of 3-day post-MI heart. On the other hand, while blockade of AT(1)R significantly ameliorated apoptotic changes in 1-day post-MI border zone, blockade of JAK2 increased apoptosis. CONCLUSIONS: The study provides compelling evidence in favor of the linkage of the JAK-STAT pathway with the angiotensin II autocrine loop and uncovers a mechanism by which selective activation of a set of STAT proteins underlies mobilization of the gene activation program intrinsic to post-MI remodeling. It also suggests that drugs that inhibit JAK-STAT phosphorylation may provide a new approach to modify post-MI remodeling. This needs to be confirmed in long term in vivo studies in the post-MI heart.

Angiotensin II↗
PubMed2002

Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal.

The K state, an early intermediate of the bacteriorhodopsin photocycle, contains the excess free energy used for light-driven proton transport. The energy gain must reside in or near the photoisomerized retinal, but in what form has long been an open question. We produced the K intermediate in bacteriorhodopsin crystals in a photostationary state at 100K, with 40% yield, and determined its X-ray diffraction structure to 1.43 A resolution. In independent refinements of data from four crystals, the changes are confined mainly to the photoisomerized retinal. The retinal is 13-cis,15-anti, as known from vibrational spectroscopy. The C13=C14 bond is rotated nearly fully to cis from the initial trans configuration, but the C14-C15 and C15=NZ bonds are partially counter-rotated. This strained geometry keeps the direction of the Schiff base N-H bond vector roughly in the extracellular direction, but the angle of its hydrogen bond with water 402, that connects it to the anionic Asp85 and Asp212, is not optimal. Weakening of this hydrogen bond may account for many of the reported features of the infrared spectrum of K, and for its photoelectric signal, as well as the deprotonation of the Schiff base later in the cycle. Importantly, although 13-cis, the retinal does not assume the expected bent shape of this configuration. Comparison of the calculated energy of the increased angle of C12-C13=C14, that allows this distortion, with the earlier reported calorimetric measurement of the enthalpy gain of the K state indicates that a significant part of the excess energy is conserved in the bond strain at C13.

Bacteriorhodopsins↗
PubMed2002

A protein kinase C inhibitor induces phenotypic reversion of ras-transformed pancreatic cancer cells and cooperatively blocks tumor cell proliferation with an anti- ras peptide.

PURPOSE: We have previously found that the staurosporine derivative, CGP 41 251, that has a high specificity for inhibiting protein kinase C (PKC), selectively blocks oncogenic ras-p21-induced oocyte maturation and that PKC and jun-N-terminal kinase (JNK), with which oncogenic ras-p21 directly interacts, reciprocally require each other's activation. We sought to determine whether CGP 41 251 blocks proliferation of ras-transformed mammalian cells and whether it synergistically exerts this effect with a ras-p21 peptide (residues 96-110) that interferes with the interaction of ras-p21 with JNK. METHODS: We incubated ras-transformed rat pancreatic cancer TUC-3 cells and their normal counterpart pancreatic acinar BMRPA1 cells with CGP 42 251 alone and in the presence of the ras-p21 96-110 peptide, both in pre- and post-monolayer phases and determined cell counts and morphology and, for TUC-3 cells, their ability to grow on soft agar. In the post-monolayer experiments, we also evaluated these parameters after withdrawal of these agents. RESULTS: CGP 41 251, but not its inactive analogue, CGP 42 700, blocked pre-monolayer growth and reduced post-monolayer cell counts of both TUC-3 and BMRPA1 cells (IC(50) 0.28 and 0.35 micro M, respectively). After 2 weeks of treatment, all the remaining TUC-3 cells exhibited the untransformed phenotype. Withdrawal of CGP 41 251 resulted in almost complete regrowth of the normal BMRPA1 cells while the reverted TUC-3 cells grew much more slowly. These effects were greatly enhanced by the presence of the ras-p21 96-110 peptide. CONCLUSIONS: CGP 41 251 strongly blocks growth of ras-transformed pancreatic cancer cells by causing cell death and by induction of phenotypic reversion. The enhancement of this effect by the ras-p21 96-110 peptide indicated synergy between it and CGP 41 251, allowing it to block proliferation of the transformed cells selectively. These findings suggest the possibility of using these two agents in anticancer therapy.

Antineoplastic Agents↗
PubMed2002

A dyspnea evaluation protocol for respiratory therapists: a feasibility study.

PURPOSE: We tested the feasibility of incorporating a dyspnea evaluation protocol into bedside assessments routinely performed by respiratory therapists (RTs) on mechanically ventilated patients at a university teaching hospital. METHODS: A dyspnea assessment protocol was incorporated into the RT assessments performed at 4-hour intervals on endotracheally intubated, mechanically ventilated patients in our medical and surgical intensive care units. RTs were asked to inquire of all responsive patients: "Are you feeling short of breath right now?" and, if yes, "Is your shortness of breath mild, moderate, or severe?" We analyzed 324 consecutive patient ventilator flow sheets from 77 medical and 161 surgical intensive care unit patients. RESULTS: Dyspnea scores were recorded during 1,870 of 2,539 scheduled RT patient assessments. The protocol compliance rate was 74%. Patients were sufficiently responsive to answer the protocol questions during 32.1% of the bedside assessments. Dyspnea was recorded in 11% (67/600) of those encounters. Dyspnea was described most often as mild. CONCLUSIONS: Initial implementation of a dyspnea evaluation protocol was moderately successful in prompting RTs to ask mechanically ventilated patients whether they felt short of breath during scheduled bedside visits. A rapid bedside evaluation for dyspnea may prove useful in evaluating the effect on patient distress of implementing protocols designed to optimize ventilator settings or the use of sedating drugs during mechanical ventilation. By this approach RTs may also be able to promote a patient-centered approach to managing respiratory failure in the intensive care unit.

Dyspnea↗