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D F Smith

Publications and source records attributed to D F Smith.

At least 37 records · Page 2Linked to original sources
PubMed2001

Quantitative radioluminography of serotonin uptake sites in the porcine brain.

The regional density of serotonin uptake sites in porcine brain was determined by quantitative radioluminography. Brain cryostat sections 30 microm thick were cut in the sagittal plane and were incubated with [3H]citalopram for selective labeling of serotonin uptake sites. The autoradiograms were quantified using tritium-sensitive radioluminography. The apparent affinity (K(D)) of [3H]citalopram for its binding sites in various brain regions ranged from 2.3-5.6 nM. The density of serotonin uptake sites was highest (200-300 fmol/mg tissue) in the amygdala, superior colliculus, and substantia nigra. Intermediate binding (100 fmol/mg tissue) was present in the dorsomedial thalamus, basal ganglia, and entorhinal cortex. Traces of specific binding (10 fmol/mg tissue) were detected in the neocortex and cerebellar cortex. The findings show that the anatomic distribution of serotonin uptake sites in the porcine brain is similar to that reported in other mammals. The density was close to that reported in human brain and in rat brain.

Animals↗
PubMed2001

GPI-anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida.

The plasma membranes of the divergent eukaryotic parasites, Leishmania and Trypanosoma, are highly specialised, with a thick coat of glycoconjugates and glycoproteins playing a central role in virulence. Unusually, the majority of these surface macro-molecules are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In mammalian cells and yeast, many GPI-anchored molecules associate with sphingolipid and cholesterol-rich detergent-resistant membranes, known as lipid rafts. Here we show that GPI-anchored parasite macro-molecules (but not the dual acylated Leishmania surface protein (hydrophilic acylated surface protein) or a subset of the GPI-anchored glycoinositol phospholipid glycolipids) are enriched in a sphingolipid/sterol-rich fraction resistant to cold detergent extraction. This observation is consistent with the presence of functional lipid rafts in these ancient, highly polarised organisms.

Animals↗
PubMed2001

PET neuroimaging of clomipramine challenge in humans: focus on the thalamus.

PET neuroimaging of serotonin responsivity relied previously mainly on fenfluramine, but that drug has been withdrawn from the market. Therefore, we determined whether clomipramine, which stimulates serotonergic mechanisms by inhibiting serotonin reuptake, has reliable effects in the healthy human brain as measured by [15O]H2O PET. The clomipramine challenge markedly reduced the relative rate of blood flow in the selected region of interest, namely the dorsomedial nucleus of the thalamus, a limbic region rich in serotonin uptake sites. These findings show similarities between effects of fenfluramine and clomipramine in the healthy human brain, and support the use of the clomipramine challenge in conjunction with PET for studying cerebral serotonergic mechanisms.

Aged↗
PubMed2001

The growth of chondrocytes into a fibronectin-coated biodegradable scaffold.

Porous scaffolds made from a biodegradable copolymer of trimethylene carbonate and glycolide were evaluated for tissue-engineered medical products. We examined the scaffold coated with cell adhesion protein and fibronectin and cultured under a dynamic mixing condition to enhance the growth of chondrocytes. Our hypothesis was that the combination of coating and dynamic mixing would be beneficial to the viability of the chondrocytic cells. Fibronectin was selected as the model protein because of its availability and routine assaying methods. Sterile samples of scaffolds of about 1 mm in thickness were coated with fibronectin at 37 degrees C for 1.5 h. Four groups of scaffolds were used: uncoated static or dynamic, and coated static or dynamic. Scaffold samples were placed in either a Petri dish or a spinner flask (static vs. dynamic groups) after inoculation with rat chondrocytes of an initial cell density of 1.29 x 10(5) cell/mL. After 7, 14, 21, and 28 days, each sample was fixed, embedded, and sectioned at 5 micro thickness. The sections were double-label immunostained using antibodies against cellular fibronectin synthesized by adherent cells as a measure of cell viability. A Hoechst 33258 nuclear stain was used to measure the number of cells attached to the scaffold at each time interval. The slides were examined using a fluorescence microscope to determine the cell ingrowth. At least 25 fields/treatment group (except the 7 day group) were measured. The data showed that cell in-growths into the porous scaffolds were higher at all time periods for the coated dynamic group than those for the other three groups.

Animals↗
PubMed2001

Overexpression of the FK506-binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates.

Many New World primates have high circulating levels of cortisol to compensate for the expression of glucocorticoid receptors (GRs) with low activity. Recent work in squirrel monkeys has suggested that this may be due to either the expression of GRs that are transcriptionally incompetent or the expression of an FK506-binding immunophilin that inhibits GR binding. The goal of this study was to resolve this controversy by determining the molecular basis of glucocorticoid resistance not only in species of squirrel monkeys but also in other glucocorticoid-resistant New World primates. First, the transcriptional activity of the GR from the Bolivian squirrel monkey was compared to that of the human GR. Incubation of COS-7 cells transfected with the squirrel monkey GR with 10 nM dexamethasone resulted in a robust stimulation of MMTV-luciferase activity (up to 260-fold), which was similar in magnitude to that achieved with the human GR. Second, the effect of FK506 on GR binding was determined in cytosol from cells from two species of squirrel monkeys as well as glucocorticoid-resistant cotton-top tamarins and owl monkeys. Incubation with 10 microM FK506 increased GR binding by at least 4-fold in cytosol from cells of each of the New World primates but had no effect on GR binding in cytosol from human WI-38 VA13 cells. Third, Western blots showed elevated expression of FKBP51 in New World primate cells and liver samples from two squirrel monkey species. On the other hand, the levels of FKBP52 were significantly lower in cells and liver from New World primates. The sequences of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey are closely related and share differences from the human, rhesus monkey, mouse, and lemur FKBP51 sequences in the same 18 positions. Fourth, the relative activities of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey were determined in cytosol mixing and GR transactivation studies and showed that FKBP51 from each of these primates was a potent inhibitor of GR activity. These results indicate that the elevated expression of FKBP51 contributes to glucocorticoid resistance in three New World primate genera.

Amino Acid Sequence↗
PubMed2001

Secretion of the novel Trichinella protein TSJ5 by T. spiralis and T. pseudospiralis muscle larvae.

The Trichinella tsJ5 gene is preferentially expressed in muscle larvae of Trichinella spiralis and encodes a novel protein. Previous observations have shown tsJ5 to be expressed at higher levels in encapsulating species than in non-encapsulating species and down-regulation of gene expression in T. pseudospiralis to be correlated with a lower protein abundance in the muscle larva of this species. In the present study we have determined the full-length cDNA sequence of the tsJ5 homologue in T. pseudospiralis (tpJ5). Antigens recognised by an anti-J5 antibody are found on the cuticular surface of both T. spiralis and T. pseudospiralis muscle larvae, as well as in the body wall muscle. We show that both the TSJ5 and TPJ5 proteins are found in the excretory/secretory fractions collected from muscle larva cultured in vitro and that despite the absence of a typical N-terminal signal sequence, secretion of pTSJ5 is mediated through the classical ER/Golgi secretory pathway.

Amino Acid Sequence↗
PubMed2001

Identification and characterisation of a RAD51 gene from Leishmania major.

The RAD51 gene is a homologue of Escherichia coli recA which plays a central role in homologous recombination and DNA repair. This paper describes the identification of the RAD51 gene from the trypanosomatid parasite Leishmania major. The LmRAD51 gene codes for a 377 amino acid polypeptide with a predicted molecular mass of 41259 Da that is highly homologous to the Rad51 family of proteins. Recombinant L. major Rad51 protein (LmRad51) was over-expressed in a bacterial expression system, purified to homogeneity and shown to bind DNA and exhibit DNA-stimulated ATPase activity, consistent with previously reported biochemical characteristics of Rad51 protein. Although LmRad51 expression is below the level of detection in exponentially growing cultures of Leishmania, high levels of LmRad51 mRNA and protein expression can be detected following exposure to the DNA-damaging agent phleomycin. LmRAD51 is one of the first examples of a DNA damage-inducible gene to be characterised in Leishmania, and will be invaluable in studying the contribution of homologous recombination to Leishmania virulence.

Adenosine Triphosphatases↗
PubMed2001

Synthesis and evaluation of racemic [(11)C]NS2456 and enantiomers as selective serotonin reuptake radiotracers for PET.

Positron emission tomography (PET) radiotracers are needed for quantifying serotonin uptake sites in the living brain. Therefore, we evaluated a new selective serotonin reuptake inhibitor, NS2456, to determine whether it is suited for use in PET. Racemic NS2456 [(1RS,5SR)-8-methyl-3-[4-trifluoromethoxyphenyl]-8-azabicyclo [3.2.1]oct-2-ene] and its N-demethylated analog, racemic NS2463, selectively inhibited serotonin uptake in rat brain synaptosomes; their IC(50) values were 3000-fold lower for [(3)H]serotonin than for either [(3)H]dopamine or [(3)H]noradrenaline. The enantiomers of NS2463 were also potent inhibitors of serotonin uptake in vitro, but they failed to show stereoselectivity. Racemic NS2463 as well as its enantiomers were radiolabelled by N-methylation with C-11, yielding [(11)C]NS2456 for use in PET of the living porcine brain. The compounds crossed the blood-brain barrier rapidly and accumulated preferentially in regions rich in serotonin uptake sites (e.g., brainstem, subthalamus and thalamus). However, their binding potentials were relatively low and no stereoselectivity was found. Thus, neither racemic [(11)C]NS2456 nor its [(11)C]-labelled enantiomers are ideal for PET neuroimaging of neuronal serotonin uptake sites.

Animals↗
PubMed2001

[14C]Serotonin uptake and [O-methyl-11C]venlafaxine kinetics in porcine brain.

As part of our program of developing PET tracers for neuroimaging of psychotropic compounds, venlafaxine, an antidepressant drug, was evaluated. First, we measured in vitro rates of serotonin uptake in synaptosomes prepared from selected regions of porcine brain. Then, we determined the pharmacokinetics of venlafaxine, [O-methyl-11C]-labeled for PET. Synaptosomal studies showed that the active uptake of [14C]5-HT differed markedly between brain regions, with highest rates in hypothalamus, raphé region, and thalamus, and lowest rates in cortex and cerebellum. PET studies showed that the unidirectional rate of uptake of [O-methyl-11C]venlafaxine from blood to brain was highest in the hypothalamus, raphé region, thalamus and basal ganglia and lowest in the cortex and cerebellum. Under normal physiological conditions, the capillary permeability-surface area (PS) product for [O-methyl-11C]venlafaxine could not be estimated, because of complete flow-limitation of the cerebral uptake. Nevertheless, a correlation occurred between the apparent partition volume of the radiotracer and the rate of active uptake of 5-HT in selected regions of the porcine brain. During hypercapnia, limitations of blood-brain transfer were observed, giving PS-products for water that were only ca. 50% higher than those of venlafaxine. Thus, under normal physiological conditions, the rate of uptake of venlafaxine from blood into brain is completely flow-limited.

Animals↗
PubMed2001

Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania.

The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1, HASPA2 and HASPB, encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 and SHERP2, coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild-type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo. Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement-mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.

Animals↗
PubMed2001

Enhanced protection from renal ischemia-reperfusion [correction of ischemia:reperfusion] injury with A(2A)-adenosine receptor activation and PDE 4 inhibition.

BACKGROUND: We previously demonstrated in rats and mice that agonists of A(2A)-adenosine receptors (A(2A)-ARs) reduce renal injury following ischemia-reperfusion. We now extend these studies and examine the effects of ATL-146e (formerly DWH-146e), an A(2A)-AR agonist, and rolipram, a type IV phosphodiesterase (PDE 4) inhibitor, on murine renal injury following ischemia-reperfusion. METHODS: C57BL/6 mice were treated with rolipram, ATL-146e, or both compounds combined and were subjected to renal ischemia for 32 minutes and reperfusion for 24 to 48 hours. In vitro studies were performed on suspended and adhering human neutrophils. RESULTS: Continuous delivery of rolipram or ATL-146e during reperfusion reduced renal injury in a dose-dependent manner. Maximal protection was observed when ATL-146e was infused for six hours during reperfusion. Elevated plasma creatinine and myeloperoxidase activity produced by ischemia-reperfusion were reduced by rolipram (0.1 ng/kg/min) and ATL-146e (10 ng/kg/min) by up to approximately 60% and 70%, respectively. Co-infusion of both compounds produced a maximum reduction of plasma creatinine of approximately 90% and myeloperoxidase activity. In vitro studies on suspended and adhering human neutrophils demonstrated that selective stimulation of A(2A)-ARs by ATL-146e increased cAMP accumulation, reduced oxidative activity of activated neutrophils, and decreased activated neutrophil adherence. These responses were potentiated by rolipram. CONCLUSIONS: We conclude that the combined infusion of ATL-146e and rolipram leads to enhanced renal tissue protection from ischemia-reperfusion by mechanisms that may include reduced neutrophil adherence/recruitment and release of reactive oxygen species.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Negative emotions and coronary heart disease: causally related or merely coexistent? A review.

Negative emotions have been claimed to be a cause of coronary heart disease (CHD) as well as a consequence of cardiovascular disorders. Early case studies of cardiac disorders of soldiers in battle drew attention to the possibility that strong negative emotional states could cause CHD. Subsequent reports of reactions to natural disasters supported the notion that intense negative emotions could precipitate somatic disorders such as CHD. Since then, numerous studies have investigated relations between negative emotions and CHD. Over the years, retrospective studies have found, for example, that negative emotions are often present before the occurrence of CHD. Cross-sectional studies have indicated that symptoms of depression and anxiety are often present in CHD patients. Prospective studies have shown that the likelihood of CHD tends to be higher for people with negative emotions than for those without them. The main symptoms of negative emotional states that seem to be most closely associated with CHD are nervousness, getting easily upset, feeling fatigue, being indecisive, having sleep disturbances, being usually worried about something, and feeling that others would be better off if oneself were dead. Although the findings appear to support the notion of causal connections between negative emotions and CHD, they fail to provide conclusive proof of such relations. An alternative explanation that could also account for the findings is simply that negative emotions and CHD often coexist.

Affective Symptoms↗
PubMed2001

Prediction of tissue survival after middle cerebral artery occlusion based on changes in the apparent diffusion of water.

OBJECT: In this study the authors tested the hypothesis that the estimate of the apparent diffusion coefficient (ADC) of water is a reliable pathophysiological index of the viability of ischemic brain tissue. METHODS: Cerebral blood flow (CBF) and the cerebral metabolic rates of oxygen and glucose (CMRO2 and CMRglc, respectively) were measured using positron emission tomography (PET) scanning before and after permanent middle cerebral artery occlusion (MCAO) or reperfusion in pigs. The ADC value, which was measured using diffusion-weighted magnetic resonance (DW MR) imaging was compared with physiological variables obtained by PET scanning and with histological findings. After both permanent MCAO and reperfusion, the decrease in the ADC was significantly correlated with decrease in the CMRO2 and CMRglc. The infarction coincided with a CMRO2 threshold of 50% of the value measured on the contralateral side. Thus, an ADC value of 80% or 75% of the contralateral value reflected the CMRO2 threshold after permanent MCAO or reperfusion, respectively. On DW MR images, lesions with ADC values above 80% of the contralateral value are potentially reversible until 6 hours after MCAO, whereas lesions with ADC values below 75% of the contralateral value are irreversible as early as 2 hours after MCAO. CONCLUSIONS: The ADC of water provides a reliable pathophysiological index for tailoring therapy to the condition of individual stroke patients in clinical practice.

Animals↗
PubMed2000

Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis.

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4(+) T cells producing IFN-gamma, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-gamma-producing CD8(+) T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.

Animals↗
PubMed2000

Acylation-dependent protein export in Leishmania.

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

Acylation↗
PubMed2000

Poly(A)-binding protein I of Leishmania: functional analysis and localisation in trypanosomatid parasites.

Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional. Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript stability is a major factor controlling stage-specific gene expression. Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (Lm PAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression. lmpab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids. Lm PAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo. It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus. Lm PAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae. These properties are indicative of functional divergence in vivo.

Amino Acid Sequence↗