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Biomedical subjects

E W Taylor

Publications and source records attributed to E W Taylor.

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

High affinity binding of brain myosin-Va to F-actin induced by calcium in the presence of ATP.

Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca(2+) in a highly cooperative manner. We demonstrate here that Ca(2+) also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca(2+)-induced cosedimentation. The Ca(2+)-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca(2+) was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca(2+)-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5'-O-(thiotriphosphate) is induced by micromolar concentrations of Ca(2+). Since Ca(2+) regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.

Actin Cytoskeleton↗
PubMed2001

Protein synthesis and specific dynamic action in crustaceans: effects of temperature.

Temperature influences the specific dynamic action (SDA), or rise in oxygen uptake rate after feeding, in eurythermal and stenothermal crustaceans by changing the timing and the magnitude of the response. Intra-specific studies on the eurythermal crab, Carcinus maenas, show that a reduction in acclimation temperature is associated with a decrease in SDA magnitude, resulting from an increase in SDA duration but a decrease in peak factorial scope (the factorial rise in peak SDA over prefeeding values). Inter-specific feeding studies on stenothermal polar isopods revealed marked differences in SDA response between the Antarctic species, Glyptonotus antarcticus and the Arctic species, Saduria entomon. Compared to S. entomon held at 4 and 13 degrees C, the SDA response in G. antarcticus held at 1 degrees C was characterised by a lower absolute oxygen uptake rate at peak SDA and an extended SDA duration. At peak SDA, whole animal rates of protein synthesis increased in proportion to the postprandial increase in oxygen uptake rate in the Antarctic and the Arctic species. Rates of oxygen uptake plotted against whole animal rates of protein synthesis gave similar relationships in both isopod species, indicating similar costs of protein synthesis after a meal, despite their differences in SDA response and thermal habitat.

Animals↗
PubMed2001

The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa.

Lungfishes (Dipnoi) occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood PO(2) per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution.

Anemia↗
PubMed2001

Effects of sublethal ammonia exposure on swimming performance in rainbow trout (Oncorhynchus mykiss).

Adult trout Oncorhynchus mykiss fitted with a dorsal aortic catheter were exposed to 288+/-15 micromol l(-1) (mean +/- S.E.M.) total ammonia for 24h in water at a pH of 8.39+/-0.02, while swimming at a speed equivalent to 0.75 bodylengths s(-1) (BLs(-1)) in a Brett-type tunnel respirometer. The fish were then exposed to stepwise increments in swimming speed (0.25 BLs(-1) every 30 min) until exhaustion. Measurements of oxygen uptake (M(O2)) and plasma total ammonia levels and pH were made at each speed. Control trout were treated identically but without exposure to ammonia. Ammonia exposure caused an increase in plasma total ammonia level to 436+/-34 micromol l(-1), compared to 183+/-30 micromol l(-1)in control animals (N=6). A significant reduction in total plasma ammonia level was found in both groups during exercise, despite a large negative concentration gradient in those exposed to an elevated concentration of ammonia in water, which may indicate an active excretory process. The overall increase in plasma ammonia levels in exposed trout was associated with a significant reduction in critical swimming speed (U(crit)) to 1.61+/-0.17BL s(-1) from 2.23+/-0.15BL s(-1) in control animals. Ammonia-exposed trout had a significantly higher maintenance metabolic rate (MMR) than control fish, when estimated as the y-intercept of the relationship between swimming speed and M(O2). Active metabolic rate (AMR, maximum M(O2) as measured at U(crit)) was significantly lower in ammonia-exposed animals, leading to a profound reduction in factorial aerobic scope (AMR/MMR). Reduced U(crit) was also linked to a reduction in maximum tailbeat frequency. Calculation of membrane potentials (E(M)) in the white muscle of fish swum to U(crit) revealed a significant partial depolarisation of white muscle in ammonia-exposed fish. This may have prevented white muscle recruitment and contributed to the reduced maximum tailbeat frequency and overall impairment of swimming performance in the ammonia-exposed fish.

Ammonia↗
PubMed2001

Autonomic control of heart rate during forced activity and digestion in the snake Boa constrictor.

Reptiles, particularly snakes, exhibit large and quantitatively similar increments in metabolic rate during muscular exercise and following a meal, when they are apparently inactive. The cardiovascular responses are similar during these two states, but the underlying autonomic control of the heart remains unknown. We describe both adrenergic and cholinergic tonus on the heart during rest, during enforced activity and during digestion (24-36 h after ingestion of 30 % of their body mass) in the snake Boa constrictor. The snakes were equipped with an arterial catheter for measurements of blood pressure and heart rate, and autonomic tonus was determined following infusion of the beta-adrenergic antagonist propranolol (3 mg kg(-1)) and the muscarinic cholinoceptor antagonist atropine (3 mg kg(-1)). The mean heart rate of fasting animals at rest was 26.4+/-1.4 min(-1), and this increased to 36.1+/-1.4 min(-1) (means +/- S.E.M.; N=8) following double autonomic block (atropine and propranolol). The calculated cholinergic and adrenergic tones were 60.1+/-9.3 % and 19.8+/-2.2 %, respectively. Heart rate increased to 61.4+/-1.5 min(-1) during enforced activity, and this response was significantly reduced by propranolol (maximum values of 35.8+/-1.6 min(-1)), but unaffected by atropine. The cholinergic and adrenergic tones were 2.6+/-2.2 and 41.3+/-1.9 % during activity, respectively. Double autonomic block virtually abolished tachycardia associated with enforced activity (heart rate increased significantly from 36.1+/-1.4 to 37.6+/-1.3 min(-1)), indicating that non-adrenergic, non-cholinergic effectors are not involved in regulating heart rate during activity. Blood pressure also increased during activity. Digestion was accompanied by an increase in heart rate from 25.6+/-1.3 to 47.7+/-2.2 min(-1) (N=8). In these animals, heart rate decreased to 44.2+/-2.7 min(-1) following propranolol infusion and increased to 53.9+/-1.8 min(-1) after infusion of atropine, resulting in small cholinergic and adrenergic tones (6.0+/-3.5 and 11.1+/-1.1 %, respectively). The heart rate of digesting snakes was 47.0+/-1.0 min(-1) after double autonomic blockade, which is significantly higher than the value of 36.1+/-1.4 min(-1) in double-blocked fasting animals at rest. Therefore, it appears that some other factor exerts a positive chronotropic effect during digestion, and we propose that this factor may be a circulating regulatory peptide, possibly liberated from the gastrointestinal system in response to the presence of food.

Adrenergic beta-Antagonists↗
PubMed2001

Evidence of a functional role in lung inflation for the buccal pump in the agamid lizard, Uromastyx aegyptius microlepis.

This study has demonstrated that the agamid desert lizard Uromastyx aegyptius microlepis ventilates its lungs both with a triphasic, thoracic aspiratory pump and by gulping air, using a buccal pump. These two mechanisms never occur simultaneously because bouts of buccal pumping are always initiated after the passive expiration that terminates a thoracic breath. Lung inflation arising from thoracic and buccal ventilation was confirmed by direct recording of volume changes using a whole-body plethysmograph. This observation was further confirmed by mechanical separation of the inflationary pressures associated with these two breathing mechanisms, enabling the effects of lung inflation on buccal breathing to be observed. This revealed that the buccal pump is influenced by a powerful Hering-Breuer-type reflex, further confirming its role in lung inflation. Bilateral thoracic vagotomy tended to increase the variance of the amplitude and duration of the breaths associated with the aspiration pump and abolished the effects of lung inflation on the buccal pump. Uromastyx has vagal afferents from pulmonary receptors that respond to changes in lung volume and appear not to be sensitive to CO2. This study describes two lung-inflation mechanisms (an amphibian-like buccal pump and a mammalian-like aspiration pump) in a single extant amniote, both of which are subject to vagal feedback control.

Animals↗
PubMed2001

Lactate sequestration in the carapace of the crayfish Austropotamobius pallipes during exposure in air.

When held in air for up to 24 h, crayfish accumulated Ca(2+) and Mg(2+) in their haemolymph in direct proportion to raised levels of lactate. K(+) levels were highly variable, with elevated levels associated with morbidity. Lactate accumulation in the haemolymph was reflected in proportional increases in lactate levels in the carapace and muscle. Pieces of carapace incubated in saline containing elevated levels of lactate accumulated lactate to up to half the dissolved concentration. Measured levels in the carapace, relative to its water content, implied that lactate accumulated in the carapace in a combined form, possibly complexed to calcium. The exoskeleton seems to provide a reserve of buffering capacity and a sink for lactate during anaerobic metabolism. A similar mechanism has been identified in pond turtles.

Air↗
PubMed2000

Nutrition, HIV, and drug abuse: the molecular basis of a unique role for selenium.

HIV-infected injection drug users (IDUs) often suffer from serious nutritional deficiencies. This is a concern because plasma levels of micronutrients such as vitamin B12, zinc, and selenium have been correlated with mortality risk in HIV-positive populations. Injection drug use also increases lipid peroxidation and other indicators of oxidative stress, which, combined with antioxidant deficiencies, can stimulate HIV-1 replication through activation of NF-kappaB transcription factors, while weakening immune defenses. As detailed herein, these prooxidant stimuli can also increase the pathogenic effects of HIV-1 by another mechanism, involving viral selenoproteins. Overlapping the envelope coding region, HIV-1 encodes a truncated glutathione peroxidase (GPx) gene (see #6 in reference list). Sequence analysis and molecular modeling show that this viral GPx (vGPx) module has highly significant structural similarity to known mammalian GPx, with conservation of the catalytic triad of selenocysteine (Sec), glutamine, and tryptophan. In addition to other functions, HIV-1 vGPx may serve as a negative regulator of proviral transcription, by acting as an NF-kappaB inhibitor (a known property of cellular GPx). Another potential selenoprotein coding function of HIV-1 is associated with the 3' end of the nef gene, which terminates in a conserved UGA (potential Sec) codon in the context of a sequence (Cys-Sec) identical to the C-terminal redox center of thioredoxin reductase, another cellular regulator of NF-kappaB. Thus, in combination with known cellular mechanisms involving Se, viral selenoproteins may represent a unique mechanism by which HIV-1 monitors and exploits an essential micronutrient to optimize its replication relative to the host.

Amino Acid Sequence↗
PubMed2000

Molecular modeling and in vitro activity of an HIV-1-encoded glutathione peroxidase.

Based on theoretical evidence, it has been proposed that HIV-1 may encode several selenoprotein modules, one of which (overlapping the env gp41-coding region) has highly significant sequence similarity to the mammalian selenoprotein glutathione peroxidase (GPx; EC ). The similarity score of the putative HIV-1 viral GPx homolog relative to an aligned set of known GPx is 6.3 SD higher than expected for random sequences of similar composition. Based on that alignment, a molecular model of the HIV-1 GPx was constructed by homology modeling from the bovine GPx crystal structure. Despite extensive truncation relative to the cellular GPx gene, the structural core and the geometry of the catalytic triad of selenocysteine, glutamine, and tryptophan are well conserved in the viral GPx. All of the insertions and deletions predicted by the alignment proved to be structurally feasible. The model is energetically favorable, with a computed molecular mechanics strain energy close to that of the bovine GPx structure, when normalized on a per-residue basis. However, considering the remote homology, this model is intended only to provide a working hypothesis allowing for a similar active site and structural core. To validate the theoretical predictions, we cloned the hypothetical HIV-1 gene and found it to encode functional GPx activity when expressed as a selenoprotein in mammalian cells. In transfected canine kidney cells, the increase in GPx activity ranged from 21% to 43% relative to controls (average 30%, n = 9, P < 0.0001), whereas, in transfected MCF7 cells, which have low endogenous GPx activity, a near 100% increase was observed (average 99%, n = 3, P < 0.05).

Amino Acid Sequence↗
PubMed2000

High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry.

We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS(3)). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the beta-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1beta, to build an FGF-2 homology model with a backbone error of 4.8 A (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation.

Animals↗
PubMed2000

Exposure of brown trout, Salmo trutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential.

Brown trout acclimated to soft water and exposed for 96 h to a sub-lethal concentration of copper at low pH (0.08 micromol l(-1) Cu, pH 5) have a lower critical swimming speed than fish from copper-free water at neutral pH. This loss of performance is not due to difficulties in oxygen transfer resulting from gill damage since arterial oxygen and carbon dioxide levels remain unaffected. Both red and white muscle showed some metabolic disruptions consistent with local hypoxia, namely a high lactate concentration at rest and, in the white muscle, depletion of glycogen and phosphocreatine. However, a putative role of increased blood viscosity following haematological changes in reducing the supply of oxygen to the tissues is not supported by the current study. Haematocrit, haemoglobin and plasma protein concentrations were not affected by this treatment and a lack of further change in variables such as lactate at the onset of exercise led one to look for an alternative explanation for the effects of copper and low pH upon tissue metabolites. Ammonia concentration, both in the plasma and muscles, is significantly higher in trout exposed to copper and low pH. Ammonia plays a role in the regulation of a number of metabolic pathways and could contribute to the altered metabolic status of these fish. In addition, ammonium ions are known to cause electrophysiological disruptions, particularly the displacement of K(+) in ion exchange mechanisms that could lead to the observed loss of swimming performance. Using the measured distribution of ammonia between intracellular and extracellular compartments to estimate membrane potential of resting muscle, a significant depolarisation is predicted in both red and white muscle of fish exposed to copper and low pH.

Algorithms↗
PubMed2000

The resting membrane potential of white muscle from brown trout (Salmo trutta) exposed to copper in soft, acidic water.

Previously, the distribution of ammonia between the intracellular and extracellular compartments has been used to predict a significant depolarisation of the resting membrane potential (E(M)) of white muscle from brown trout (Salmo trutta) exposed to a sub-lethal combination of copper and low pH. However, this prediction is based upon two assumptions (i) a relatively high membrane permeability for the ammonium ion with respect to that for ammonia gas and (ii) that this is unaltered by exposure to copper and low pH. Since there is conflicting evidence in the literature of the validity of these assumptions, in the present study E(M) was directly measured in white muscle fibres of trout exposed to copper and low pH (E(M)=-52.2+/-4.9 mV) and compared with that of unexposed, control animals (E(M)=-86.5+/-2.9 mV) (means +/- s.e.m., N=6). In confirming the predicted depolarisation, these data support the hypothesis of electrophysiological impairment as a factor in the reduction in the swimming performance of trout exposed to these pollutants. In addition, the results of this study support the role of a significant permeability of the muscle membrane to NH(4)(+) in determining the distribution of ammonia in fish.

Acids↗
PubMed1999

A structural change in the kinesin motor protein that drives motility.

Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.

Adenosine Triphosphatases↗
PubMed1999

Lung deflation stimulates fictive ventilation in decerebrated and unidirectionally ventilated toads.

We describe how the degree of lung inflation and hypercapnia influenced fictive ventilation in five toads (Bufo marinus) that were decerebrated and paralysed with roccuronium. Both lungs were unidirectionally ventilated and the degree of lung inflation was determined by controlling the outflow resistance of these catheters, while ventilatory motor output was assessed on the basis of nervous activity in the mandibular branch of the Vth cranial nerve. The pattern of the recorded activity ('fictive ventilation') resembled the ventilatory patterns previously described for conscious toads. Increasing the fraction of CO2 in the gas mixture used for unidirectional ventilation from 0.00 to 0.05 stimulated fictive breathing. Fictive ventilation was also greatly stimulated, at all CO2 levels, by reduced lung volume, while complete inflation of the lungs abated fictive ventilation at all levels of CO2. Stimulation of CO2 sensitive chemoreceptors and pulmonary stretch receptors appear to have interactive effects on the central generation of ventilatory output in toads.

Animals↗
PubMed1999

Hepatitis C virus encodes a selenium-dependent glutathione peroxidase gene. Implications for oxidative stress as a risk factor in progression to hepatocellular carcinoma.

AIM: Using structural bioinformatics methods, the aim is to assess the hypothesis that hepatitis C virus (HCV) encodes a glutathione peroxidase (GPx) gene in an overlapping reading frame, linking HCV expression and pathogenesis to the Se status and dietary oxidant/Antioxidant balance of the host. METHODS: The putative HCV GPx gene was identified by searching viral sequence databases, using conserved GPx active site sequences as probes, giving particular weight to the UGA (selenocysteine) codon. Multiple sequence alignments were generated and analyzed to validate the sequence similarity, and to establish the degree of conservation of the identified genomic features in HCV. Molecular modeling was used to assess the structural feasibility of the proposed homology. RESULTS: The GPx homology region overlaps the NS4 gene, and is well conserved in HCV. The sequence similarity of the conserved active site regions to a set of known GPx is high (4 to 6 SD greater than expected for similar random sequences). The computed strain energy of a molecular model of the HCV GPx is energetically favorable, comparable to the bovine GPx structure. CONCLUSIONS: By linking HCV replication and pathogenesis to the Se status and dietary oxidant/antioxidant balance of the host, the existence of a viral GPx gene could help to explain why HCV disease progression is accelerated by oxidant stresses such as alcoholism and iron overload.

Amino Acid Sequence↗