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Biomedical subjects

S V Smith

Publications and source records attributed to S V Smith.

At least 19 recordsLinked to original sources
PubMed2002

Distribution and significance of small, artificial water bodies across the United States landscape.

At least 2.6 million small, artificial water bodies dot the landscape of the conterminous United States; most are in the eastern half of the country. These features account for approximately 20% of the standing water area across the United States, and their impact on hydrology, sedimentology, geochemistry, and ecology is apparently large in proportion to their area. These features locally elevate evaporation, divert and delay downstream water flow, and modify groundwater interactions. They apparently intercept about as much eroded soil as larger, better-documented reservoirs. Estimated vertical accretion rates are much higher, hence, inferred sedimentary chemical reactions must be different in the small features than in larger ones. Finally, these features substantially alter the characteristics of aquatic habitats across the landscape.

Conservation of Natural Resources↗
PubMed2001

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding.

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.

Acids↗
PubMed2001

Nitrate esters as nitric oxide donors: SS-nitrates.

[structure: see text]. The important biological secondary messenger NO can be generated from exogenous nitrovasodilators and NO donors. Nitrate esters are nitrovasodilators and NO mimetics, believed to be biotransformed to NO in vivo. On the basis of a mechanistic hypothesis, nitrates have been synthesized that release NO at significant rates in neutral aqueous solution in the presence only of added thiol. The novel masked beta-mercaptonitrates reported (SS-nitrates), provide information on possible sulfhydryl-dependent biotransformation mechanisms for nitrates in clinical use.

Electrochemistry↗
PubMed2001

GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels.

GABA emerges as a trophic signal during rat neocortical development in which it modulates proliferation of neuronal progenitors in the ventricular/subventricular zone (VZ/SVZ) and mediates radial migration of neurons from the VZ/SVZ to the cortical plate/subplate (CP/SP) region. In this study we investigated the role of GABA in the earliest phases of neuronal differentiation in the CP/SP. GABAergic-signaling components emerging during neuronal lineage progression were comprehensively characterized using flow cytometry and immunophenotyping together with physiological indicator dyes. During migration from the VZ/SVZ to the CP/SP, differentiating cortical neurons became predominantly GABAergic, and their dominant GABA(A) receptor subunit expression pattern changed from alpha4beta1gamma1 to alpha3beta3gamma2gamma3 coincident with an increasing potency of GABA on GABA(A) receptor-mediated depolarization. GABA(A) autoreceptor/Cl(-) channel activity in cultured CP/SP neurons dominated their baseline potential and indirectly their cytosolic Ca(2+) (Ca(2+)c) levels via Ca(2+) entry through L-type Ca(2+) channels. Block of this autocrine circuit at the level of GABA synthesis, GABA(A) receptor activation, intracellular Cl(-) ion homeostasis, or L-type Ca(2+) channels attenuated neurite outgrowth in most GABAergic CP/SP neurons. In the absence of autocrine GABAergic signaling, neuritogenesis could be preserved by depolarizing cells and elevating Ca(2+)c. These results reveal a morphogenic role for GABA during embryonic neocortical neuron development that involves GABA(A) autoreceptors and L-type Ca(2+) channels.

Action Potentials↗
PubMed2001

GABA(B) receptors mediate motility signals for migrating embryonic cortical cells.

During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp, near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry, RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immuno- cytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABA(B)-R(+) cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R(+), >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

Amino Acids↗
PubMed2001

Sustainability of coastal resource use in San Quintin, Mexico.

San Quintin, Mexico, provides a useful site for integrated analyses of material fluxes and socioeconomic constraints in a geographically isolated system. Natural resource utilization on the land is dominated by groundwater exploitation for cultivation of horticulture crops (primarily tomatoes). Irrigation exceeds water recharge minus export by a factor of 6. Resource utilization in the bay is dominated by oyster culture; food for the oysters is provided by tidal exchange of bay and ocean water. Consideration of oyster respiration and system respiration suggests that the present level of aquaculture is about 40% of the sustainable level. A "physical unsustainability index" (PhUI) was developed to measure the proportional departure of utilization of the most limiting resource for sustainability: 6 on land; 0.4 in the bay. Based on PhUI and measures of economic development, we conclude that aquaculture is more viable than agriculture.

Animals↗
PubMed2001

SGN-10. Seattle Genetics.

Seattle Genetics is developing SGN-10 (BR96-SCIT), a single-chain immunotoxin (SCIT) under license from Bristol-Myers Squibb (BMS), as both a monotherapy and in combination with Taxotere (Rhône-Poulenc Rorer) for the potential treatment for cancer [308773]. SGN-10 is currently in phase I clinical trials [308773], [369809], [382807], [408963], [410348]. The compound is a follow-up to SGN-15 (BR96-DOX; BMS) and is potentially more effective and cheaper to manufacture. It delivers a bacterial toxin which is 250-times more potent than BR96-DOX and is one third the size, hence enabling tumor penetration more easily. BMS submitted an IND for SGN-10 in the first quarter of 1996 [200849], [204704]. BMS licensed the SCA-protein (single-chain antigen-binding protein) technology from Enzon in 1993 [352743]. In April 1998, Seattle Genetics completed a licensing agreement with BMS to take over development of SGN-10 [308773].

ADP Ribose Transferases↗
PubMed2000

Conformational modulation of human cytochrome P450 2E1 by ethanol and other substrates: a CO flash photolysis study.

The alcohol-inducible cytochrome P450 2E1 is a major human hepatic P450 which metabolizes a broad array of endogenous and exogenous compounds, including ethanol, low-molecular weight toxins, and fatty acids. Several substrates are known to stabilize this P450 and inhibit its cellular degradation. Furthermore, ethanol is a known modulator of P450 2E1 substrate metabolism. We examined the CO binding kinetics of P450 2E1 after laser flash photolysis of the heme-CO bond, to probe the effects of ethanol and other substrates on protein conformation and dynamics. Ethanol had an effect on the two kinetic parameters that describe CO binding: it decreased the rate of CO binding, suggesting a decrease in the protein's conformational flexibility, and increased the photosensitivity, which indicates a local effect in the active site region such as strengthening of the heme-CO bond. Other substrates decreased the CO binding rate to varying degrees. Of particular interest is the effect of arachidonic acid, which abolished photodissociation in the absence of ethanol but had no effect in the presence of ethanol. These results are consistent with a model of P450 2E1 whereby arachidonic acid binds along a long hydrophobic binding pocket and blocks exit of CO from the heme region.

Arachidonic Acid↗
PubMed2000

The reactions of myoglobin, normal adult hemoglobin, sickle cell hemoglobin and hemin with hydroxyurea.

The kinetics of the reaction of hydroxyurea (HU) with myoglobin (Mb), hemin, sickle cell hemoglobin (HbS), and normal adult hemoglobin (HbA) were determined using optical absorption spectroscopy as a function of time, wavelength, and temperature. Each reaction appeared to follow pseudo-first order kinetics. Electron paramagnetic resonance spectroscopy (EPR) experiments indicated that each reaction produced an FeNO product. Reactions of hemin and the ferric forms of HbA, HbS, and myoglobin with HU also formed the NO adduct. The formation of methemoglobin and nitric oxide-hemoglobin from these reactions may provide further insight into the mechanism of how HU benefits sickle cell patients.

Adult↗
PubMed2000

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase.

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B 1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116-134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.

Amino Acid Sequence↗
PubMed1999

Measurement of community metabolism and significance in the coral reef CO2 source-sink debate.

Two methods are commonly used to measure the community metabolism (primary production, respiration, and calcification) of shallow-water marine communities and infer air-sea CO2 fluxes: the pH-total alkalinity and pH-O2 techniques. The underlying assumptions of each technique are examined to assess the recent claim that the most widely used technique in coral reefs (pH-total alkalinity), may have provided spurious results in the past because of high rates of nitrification and release of phosphoric acid in the water column [Chisholm, J. R. M. & Barnes, D. J. (1998) Proc. Natl. Acad. Sci. USA 95, 6566-6569]. At least three lines of evidence suggest that this claim is not founded. First, the rate of nitrification required to explain the discrepancy between the two methods recently reported is not realistic as it is much higher than the rates measured in another reef system and greater than the highest rate measured in a marine environment. Second, fluxes of ammonium, nitrate, and phosphorus are not consistent with high rates of nitrification and release of phosphoric acid. Third, the consistency of the metabolic parameters obtained by using the two techniques is in good agreement in two sites recently investigated. The pH-total alkalinity technique therefore appears to be applicable in most coral reef systems. Consequently, the conclusion that most coral reef flats are sources of CO2 to the atmosphere does not need revision. Furthermore, we provide geochemical evidence that calcification in coral reefs, as well as in other calcifying ecosystems, is a long-term source of CO2 for the atmosphere.

Journal Article↗
PubMed1999

Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration.

During cortical development, embryonic neurons migrate from germinal zones near the ventricle into the cortical plate, where they organize into layers. Mechanisms that direct neuronal migration may include molecules that act as chemoattractants. In rats, GABA, which localizes near the target destination for migrating cortical neurons, stimulates embryonic neuronal migration in vitro. In mice, glutamate is highly localized near the target destinations for migrating cortical neurons. Glutamate-induced migration of murine embryonic cortical cells was evaluated in cell dissociates and cortical slice cultures. In dissociates, the chemotropic effects of glutamate were 10-fold greater than the effects of GABA, demonstrating that for murine cortical cells, glutamate is a more potent chemoattractant than GABA. Thus, cortical chemoattractants appear to differ between species. Micromolar glutamate stimulated neuronal chemotaxis that was mimicked by microM NMDA but not by other ionotropic glutamate receptor agonists (AMPA, kainate, quisqualate). Responding cells were primarily derived from immature cortical regions [ventricular zone (vz)/subventricular zone (svz)]. Bromodeoxyuridine (BrdU) pulse labeling of cortical slices cultured in NMDA antagonists (microM MK801 or APV) revealed that antagonist exposure blocked the migration of BrdU-positive cells from the vz/svz into the cortical plate. PCR confirmed the presence of NMDA receptor expression in vz/svz cells, whereas electrophysiology and Ca2+ imaging demonstrated that vz/svz cells exhibited physiological responses to NMDA. These studies indicate that, in mice, glutamate may serve as a chemoattractant for neurons in the developing cortex, signaling cells to migrate into the cortical plate via NMDA receptor activation.

Animals↗
PubMed1998

Successful treatment of transient acquired factor X deficiency by plasmapheresis with concomitant intravenous immunoglobulin and steroid therapy.

Two patients with no history of previous bleeding diatheses presented with active bleeding from multiple body sites, declining hemoglobin levels, and markedly prolonged prothrombin times (PT) and activated partial thromboplastin times (aPTT) with incomplete correction on PT mix assays. Both patients demonstrated a severe deficiency of factor X (F.X) (<1%; reference range 60-150%). F.X levels and bleeding were refractory to multiple transfusions of fresh frozen plasma (FFP) in both patients. In contrast, daily therapeutic plasma exchange (PLEX) with concomitant administration of intravenous immunoglobulin (IV IgG) and steroids produced a rapid increase in F.X levels with cessation of bleeding, followed by stabilization and normalization of F.X levels and progressive correction of coagulation times. Neither patient has demonstrated a recurrence of the bleeding tendency following discontinuation of steroid therapy. These patients had transient acquired F.X deficiency, a rare coagulopathy, which can result in a lethal bleeding diathesis. An IgG inhibitor that selectively inhibited F.X activation in Russell's viper venom or tissue factor/F.VIIa assays was demonstrated in one patient's pretreatment plasma. Previous treatment of hemorrhage in transient acquired F.X deficiency has been prothrombin complex and/or activated clotting concentrates, which can be associated with transient hypercoagulable states. This is the first reported use of PLEX in transient acquired F.X deficiency. PLEX is safe, efficacious, and rapidly restores hemostasis in this rare acquired bleeding disorder.

Adult↗
PubMed1998

188Re DD-3B6/22 Fab' for use in therapy of ovarian cancer: labelling and animal studies.

A fast and high yielding method of 188Re radiolabelling DD-3B6/22 Fab' is described. An inert atmosphere [N2(g)] and ascorbic acid was essential for preparation and storage of therapeutic levels (< or =2 GBq/mg) for up to 24 h. Immunoreactivity was greater than 75%. Pharmacokinetic studies in nu/nu mice demonstrated localisation of 188Re DD-3B6/22 Fab' was equivalent and correlated well with the behaviour observed for 99mTc DD-3B6/22 Fab' used to image ovarian cancer. Excellent stability at the target site in vivo supports the potential use of 188Re DD-3B6/22 Fab' in the therapy of ovarian cancer.

Animals↗
PubMed1998

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells.

Resting membrane potential (RMP), K+ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11-22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11-13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at approximately -70 mV and the other relatively less-polarized at approximately -40 mV. Ca2+o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a approximately Nernstian relationship between RMP and [K+]o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca2+o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K+o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU-, tetanus toxin+ cells. Differentiating neurons were estimated to contain increased K+i relative to less-polarized cells, coinciding with the developmental expression of Cs+/Ba2+-sensitive and Ca2+-dependent K+ channels. Both K+ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca2+o-dependent and tetrodotoxin-insensitive to Na+o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K+ channels and voltage-dependent Na+ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.

Animals↗
PubMed1998

GABAergic cells and signals in CNS development.

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.

Animals↗
PubMed1997

Identification of a sialidase encoded in the human major histocompatibility complex.

Mammalian sialidases are important in modulating the sialic acid content of cell-surface and intracellular glycoproteins. However, the full extent of this enzyme family and the physical and biochemical properties of its individual members are unclear. We have identified a novel gene, G9, in the human major histocompatibility complex (MHC), that encodes a 415-amino acid protein sharing 21-28% sequence identity with the bacterial sialidases and containing three copies of the Asp-block motif characteristic of these enzymes. The level of sequence identity between human G9 and a cytosolic sialidase identified in rat and hamster (28-29%) is much less than would be expected for analogous proteins in these species, suggesting that G9 is distinct from the cytosolic enzyme. Expression of G9 in insect cells has confirmed that it encodes a sialidase, which shows optimal activity at pH 4.6, but appears to have limited substrate specificity. The G9 protein carries an N-terminal signal sequence and immunofluorescence staining of COS7 cells expressing recombinant G9 shows localization of this sialidase exclusively to the endoplasmic reticulum. The location of the G9 gene, within the human MHC, corresponds to that of the murine Neu-1 locus, suggesting that these are analogous genes. One of the functions attributed to Neu-1 is the up-regulation of sialidase activity during T cell activation.

Amino Acid Sequence↗