Britain is failing to meet targets on reducing obesity.
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Female↗
Biomedical subjects
S J Smith
Publications and source records attributed to S J Smith.
The timing of synaptic vesicle endocytosis.
Alternative models to describe the endocytosis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of 20 s (24 degrees C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2+ during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
The dynamics of dendritic structure in developing hippocampal slices.
Time-lapse fluorescence confocal microscopy was used to directly visualize the formation and dynamics of postsynaptic target structures (i.e., dendritic branches and spines) on pyramidal neurons within developing tissue slices. Within a 2 week period of time, pyramidal neurons in cultured slices derived from early postnatal rat (postnatal days 2-7) developed complex dendritic arbors bearing numerous postsynaptic spines. At early stages (1-2 d in vitro), many fine filopodial protrusions on dendrite shafts rapidly extended (maximum rate approximately 2.5 microM/minute) and retracted (median filopodial lifetime, 10 min), but some filopodia transformed into growth cones and nascent dendrite branches. As dendritic arbors matured, the population of fleeting lateral filopodia was replaced by spine-like structures having a low rate of turnover. This developmental progression involved a transitional stage in which dendrites were dominated by persistent (up to 22 hr) but dynamic spiny protrusions (i.e., protospines) that showed substantial changes in length and shape on a timescale of minutes. These observations reveal a highly dynamic state of postsynaptic target structures that may actively contribute to the formation and plasticity of synaptic connections during CNS development.
Animals↗
Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons.
Norepinephrine (NE) causes an increase in the frequency of inhibitory postsynaptic potentials in CA1 pyramidal neurons in vitro. The possibility that this increase in tonic inhibition is caused by an excitatory effect on inhibitory interneurons was investigated through whole-cell recordings from pyramidal cells and both whole-cell and cell-attached patch recordings from visualized interneurons in acute slices of rat hippocampus. Adrenergic agonists caused a large increase in the frequency and amplitude of spontaneous IPSCs recorded from pyramidal cells in the presence of ionotropic glutamate receptor blockers, but they had no effect on either the frequency or the amplitude of action potential-independent miniature IPSCs recorded in tetrodotoxin. This effect was mediated primarily by an alpha adrenoceptor, although a slight beta adrenoceptor-dependent increase in IPSCs was also observed. NE caused interneurons located in all strata to depolarize and begin firing action potentials. Many of these cells had axons that ramified throughout the stratum pyramidale, suggesting that they are responsible for the IPSCs observed in pyramidal neurons. This depolarization was also mediated by an alpha adrenoceptor and was blocked by a selective alpha 1- but not a selective alpha 2-adrenoceptor antagonist. However, a slight beta adrenoceptor-dependent depolarization was detected in those interneurons that displayed time-dependent inward rectification. In the presence of a beta antagonist, NE induced an inward current that reversed near the predicted K+ equilibrium potential and was not affected by changes in intracellular Cl- concentration. In the presence of an alpha 1 antagonist, NE induced an inwardly rectifying current at potentials negative to approximately -70 mV that did not reverse (between -130 and -60 mV), characteristics similar to the hyperpolarization-activated current (lh). However, the depolarizing action of NE is attributable primarily to the alpha 1 adrenoceptor-mediated decrease in K+ conductance and not the beta adrenoceptor-dependent increase in lh. These results provide evidence that NE increases action potential-dependent IPSCs in pyramidal neurons by depolarizing surrounding inhibitory interneurons. This potent excitatory action of NE on multiple classes of hippocampal interneurons may contribute to the NE-induced decrease in the spontaneous activity of pyramidal neurons and the antiepileptic effects of NE observed in vivo.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Conformational effects in the p53 protein of mutations induced during chemical carcinogenesis: molecular dynamic and immunologic analyses.
The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen that induces the rare sentinel neoplasm angiosarcoma of the liver, has been associated with specific A-->T transversions at the first base of codons 249 and 255 of the p53 gene. These mutations result in an Arg-->Trp amino acid substitution at residue 249 and an Ile-->Phe amino acid substitution at residue 255 in a highly conserved region in the DNA-binding core domain of the p53 protein. To determine the effects of these substitutions on the three-dimensional structure of the p53 protein, we have performed molecular dynamics calculations on this core domain of the wild-type and the Trp-249 and Phe-255 mutants to compute the average structures of each of the three forms. Comparisons of the computed average structures show that both mutants differ substantially from the wild-type structure in certain common, discrete regions. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure but accessible in both mutant structures. In order to confirm this conformational shift, tumor tissue and serum from vinyl chloride-exposed individuals with angiosarcomas of the liver were examined by immunohistochemistry and enzyme-linked immunosorbent assay. Individuals with tumors that contained the p53 mutations were found to have detectable mutant p53 protein in their tumor tissue and serum, whereas individuals with tumors without mutations and normal controls did not.
Antibodies, Monoclonal↗
The role of neurophysiological investigation in traumatic brachial plexus lesions in adults and children.
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Action Potentials↗
Evidence for a role of dendritic filopodia in synaptogenesis and spine formation.
Axo-dendritic synaptogenesis was examined in live hippocampal cell cultures using the fluorescent dyes DiO to label dendrites and FM 4-64 to label functional presynaptic boutons. As the first functional synaptic boutons appeared in these cultures, numerous filopodia (up to 10 micron long) were observed to extend transiently (mean lifetime 9.5 min) from dendritic shafts. With progressively increasing numbers of boutons, there were coincident decreases in numbers of transient filopodia and increases in numbers of stable dendritic spines. Dendritic filopodia were observed to initiate physical contacts with nearby axons. This sometimes resulted in filopodial stabilization and formation of functional presynaptic boutons. These findings suggest that dendritic filopodia may actively initiate synaptogenic contacts with nearby (5-10 micron) axons and thereafter evolve into dendritic spines.
Animals↗
Potentiation of evoked vesicle turnover at individually resolved synaptic boutons.
We have studied synaptic plasticity in hippocampal cell cultures using a new imaging approach that allows unambiguous discrimination of presynaptic function at the level of single synaptic boutons. Employing a protocol designed to test for use-dependent plasticity resembling N-methyl-D-aspartate receptor-dependent long-term potentiation (NMDA-type LTP), we find that brief tetanic stimuli induce a potentiation of evoked synaptic vesicle turnover that lasts for at least 1 hr. Induction of this clearly presynaptic potentiation is blocked by putative postsynaptic glutamate receptor antagonists, suggesting that a retrograde induction signal might be involved. Potentiation appears to occur approximately equally at boutons of low and high initial release probabilities, and evidently does not involve an increase in the size of the total recycling synaptic vesicle pool.
2-Amino-5-phosphonovalerate↗
Transcranial Doppler and systemic hemodynamic studies in septic shock.
The present study outlines the relationship between cerebral and systemic hemodynamics in patients with septic shock. Sepsis is an immune mediated systemic disease in which the systemic vascular resistance (SVR) often decreases as a result of a Gram negative sepsis. The result is a hyperdynamic systemic circulation with redistribution phenomena in different organ systems. In order to study the effect of sepsis on cerebral vessels 20 patients with septic shock (12 men, 8 women, mean age 57.9 years) were subjected to both pulmonary artery catheter and transcranial Doppler (TCD) monitoring. The data were correlated to the APACHE II score and outcome. The study showed that cerebral mean and end-diastolic blood flow velocities (BFV) in the middle cerebral arteries significantly enhanced if the SVR-index decreases. In some patients a severely reduced SVRI (below 500 dynes.s/cm5.m2) was observed in combination with a downstroke latent steal phenomenon. TCD abnormalities were strongly related to disease severity and outcome. The increased BFV are explained by a mild vasospasm of the basal cerebral arteries. TCD appears to be a valuable tool to monitor the cerebral hemodynamics in these patients. They are particularly at risk for ischemic brain damage if they are subjected to therapeutic or spontaneous hyperventilation, which can potentially be detected by TCD.
APACHE↗
Synaptic vesicle recycling in synapsin I knock-out mice.
The synapsins are a family of four neuron-specific phosphoproteins that have been implicated in the regulation of neurotransmitter release. Nevertheless, knock-out mice lacking synapsin Ia and Ib, family members that are major substrates for cAMP and Ca2+/ Calmodulin (CaM)-dependent protein kinases, show limited phenotypic changes when analyzed electrophysiologically (Rosahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1995. Nature (Lond.). 375: 488-493; Rosahl, T.W., M. Geppert, D. Spillane, D., J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1993. Cell. 75:661-670; Li, L., L.S. Chin, O. Shupliakov, L. Brodin, T.S. Sihra, O. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen. 1995. Proc. Natl. Acad. Sci. USA. 92:9235-9239; see also Pieribone, V.A., O. Shupliakov, L. Brodin, S. Hilfiker-Rothenfluh, A.J. Czernik, and P. Greengard. 1995. Nature (Lond.). 375:493-497). Here, using the optical tracer FM 1-43, we characterize the details of synaptic vesicle recycling at individual synaptic boutons in hippocampal cell cultures derived from mice lacking synapsin I or wild-type equivalents. These studies show that both the number of vesicles exocytosed during brief action potential trains and the total recycling vesicle pool are significantly reduced in the synapsin I-deficient mice, while the kinetics of endocytosis and synaptic vesicle repriming appear normal.
Animals↗
Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.
Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.
Actins↗
Activation of the electrocorticogram by propofol during surgery for epilepsy.
Propofol is used widely during general anaesthesia but there has been concern that it may be implicated in provoking seizure activity. We have investigated the effects of low-dose propofol on the electrocorticogram of anaesthetized patients undergoing surgery for medically intractable epilepsy. During continuous peroperative recording of the electrocorticogram, propofol was administered in 25 mg increments until burst suppression occurred. Activation of the electrocorticogram occurred in 17 of 20 patients. There was an increase in mean spike frequency in 16, extension of spike distribution in 15 and polyphasia in 13 patients. The mean dose of propofol required to cause burst suppression was 88.2 (range 25-175) mg. We conclude that at low doses, propofol caused activation of the electrocorticogram in epileptic patients but at higher doses burst suppression was induced.
Adolescent↗
Diagnosis and treatment of status epilepticus on a neurological intensive care unit.
Status epilepticus refractory to first-line therapy is associated with a high morbidity and mortality. Correct diagnosis and adequate treatment of this condition require electrographic monitoring and anaesthetic facilities available in specialist intensive care units (ICUs). We carried out an audit of 26 patients admitted to a neurological ICU with a diagnosis of status epilepticus, to identify deficiencies in diagnosis and management prior to transfer to the ICU, and examine the effectiveness of ICU management. Or transfer, only 14 (54%) were in status epilepticus; six were in drug-induced coma or were encephalopathic, and six had pseudostatus epilepticus, of whom four had been intubated. The commonest treatments prior to transfer were benzodiazepines, chlormethiazole and phenytoin; the loading dose of phenytoin was adequate in at least 7/16 cases. All those in status epilepticus on transfer had their seizures successfully controlled, but ten required general anaesthesia with thiopentone, propofol, ketamine or midazolam. Two died--one had a severe encephalitis and the other had had a cardiac arrest prior to treatment. This study highlights deficiencies in the initial diagnosis and management of status epilepticus, the role of specialist neurological intensive care, and the importance of early referral.
Adolescent↗
Mutant p53 protein as a biomarker of chemical carcinogenesis in humans.
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Biomarkers, Tumor↗
A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization.
Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped lambda clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies.
Chromosomes, Fungal↗
Aerobic isolation of an ERG24 null mutant of Saccharomyces cerevisiae.
The ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae. We report here, however, that strains with null mutations in the ERG24 gene can grow on defined synthetic media in aerobic conditions. These sterol mutants produce ignosterol (ergosta-8,14-dienol) as the principal sterol, with no traces of ergosterol. In addition, we mapped the ERG24 gene to chromosome XIV between the MET2 and SEC2 genes. Our results indicate that ignosterol can be a suitable sterol for aerobic growth of S. cerevisiae on synthetic media and that inactivation of ERG24 is only conditionally lethal.
Aerobiosis↗
Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae.
Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae.
Aerobiosis↗
Role of spiral volumetric computed tomographic scanning in the assessment of patients with clinical suspicion of pulmonary embolism and an abnormal ventilation/perfusion lung scan.
BACKGROUND: A study was carried out to evaluate the potential place of spiral volumetric computed tomography (SVCT) in the diagnostic strategy for pulmonary embolism. METHODS: In a prospective study 249 patients with clinical suspicion of pulmonary embolism were evaluated with various imaging techniques. In all patients a ventilation/perfusion (V/Q) scan was performed. Seventy seven patients with an abnormal V/Q scan underwent SVCT. Pulmonary angiography was then performed in all 42 patients with a non-diagnostic V/Q scan and in three patients with a high probability V/Q scan without emboli on the SVCT scan. Patients with an abnormal perfusion scan also underwent ultrasonography of the legs for the detection of deep vein thrombosis. RESULTS: One hundred and seventy two patients (69%) had a normal V/Q scan. Forty two patients (17%) had a non-diagnostic V/Q scan, and in five of these patients pulmonary emboli were found both by SVCT and pulmonary angiography. In one patient, although SVCT showed no emboli, the angiogram was positive for pulmonary embolism. In one of the 42 patients the SVCT scan showed an embolus which was not confirmed by pulmonary angiography. The other 35 patients showed no sign of emboli. Thirty five patients (14%) had a high probability V/Q scan, and in 32 patients emboli were seen on SVCT images. Two patients had both a negative SVCT scan and a negative pulmonary angiogram. In one who had an inconclusive SVCT scan pulmonary angiography was positive. The sensitivity for pulmonary embolism was 95% and the specificity 97%; the positive and negative predicted values of SVCT were 97% and 97%, respectively. CONCLUSIONS: SVCT is a relatively noninvasive test for pulmonary embolism which is both sensitive and specific and which may serve as an alternative to ventilation scintigraphy and possibly to pulmonary angiography in the diagnostic strategy for pulmonary embolism.
Adult↗