bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time‐of‐flight mass spectrometry
Authors: van den Bremer Ewald T.J. , Jiskoot Wim , James Richard , Moore Geoffrey R. , Kleanthous Colin , Heck Albert J.R. , Maier Claudia S.
Published: July 2002

Nano‐electrospray ionization time‐of‐flight mass spectrometry (ESI‐MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI‐MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far‐ and near‐UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI‐MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid‐induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI‐MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn2+. Gas‐phase dissociation experiments of the deuterium‐labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas‐phase dissociation experiments of the ternary E9 DNase‐Zn2+‐Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.
Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure
Authors: Krath Britta N. , Hove‐Jensen Bjarne
Published: July 2002

Spinach 5‐phospho‐d‐ribosyl α‐1‐diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of Pi; it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. Km values for ATP and ribose 5‐phosphate are 77 and 48 μM, respectively. Gel filtration reveals a molecular mass of the native enzyme of ∼110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three‐dimensional structure has been solved, as the position and extent of helices and β‐sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.
Distance mapping of protein‐binding sites using spin‐labeled oligosaccharide ligands
Authors: Jain Nitin U. , Venot Andre , Umemoto Kimiko , Leffler Hakon , Prestegard James H.
Published: July 2002

The binding of a nitroxide spin‐labeled analog of N‐acetyllactosamine to galectin‐3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross‐peaks in the 15N heteronuclear single quantum coherence (HSQC) spectrum of full‐length galectin‐3 owing to the bound spin label is used qualitatively to idey protein residues proximate to the binding site for N‐acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross‐peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.
Effects of i‐propanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence
Authors: Zhu Keng , Jutila Arimatti , Tuominen Esa K.J. , Kinnunen Paavo K.J.
Published: July 2002

Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady‐state, time‐resolved, and stopped‐flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild‐type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so‐called “lid,” an α‐helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild‐type TLL and W89m. With increasing [iPrOH], judged by the ratio of emission intensities at 350 nm and 330 nm, the average microenvironment of the Trps in the wild‐type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time‐resolved fluorescence measurements revealed no major changes to be induced by iPrOH neither in the shorter fluorescence lifetime component (τ1 = 0.5–1.2 ns) for the wild‐type TLL nor in the longer fluorescence lifetime component (τ2 = 4.8–6.0 ns) in the wild‐type TLL and the W89m mutant. Instead, for W89m on increasing iPrOH from 25% to 50% the value for τ1 increased significantly, from 0.43 to 1.5 ns. The shorter correlation time ϕ1 of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r∞ the amplitude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped‐flow fluorescence spectroscopy measurements suggested the lid to open within ≈2 ms upon transfer of W89m into 25% iPrOH. Steady‐state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.
In search for more accurate alignments in the twilight zone
Authors: Jaroszewski Lukasz , Li Weizhong , Godzik Adam
Published: July 2002

A major bottleneck in comparative modeling is the alignment quality; this is especially true for proteins whose distant relationships could be reliably recognized only by recent advances in fold recognition. The best algorithms excel in recognizing distant homologs but often produce incorrect alignments for over 50% of protein pairs in large fold‐prediction benchmarks. The alignments obtained by sequence–sequence or sequence–structure matching algorithms differ significantly from the structural alignments. To study this problem, we developed a simplified method to explicitly enumerate all possible alignments for a pair of proteins. This allowed us to estimate the number ofsignificantly differentalignments for a given scoring method that score better than the structural alignment. Using several examples of distantly related proteins, we show that for standard sequence–sequence alignment methods, the number of significantly different alignments is usually large, often about 1010alternatives. This distance decreases when the alignment method is improved, but the number is still too large for the brute force enumeration approach. More effective strategies were needed, so we evaluated and compared two well‐known approaches for searching the space of suboptimal alignments. We combined their best features and produced a hybrid method, which yielded alignments that surpassed the original alignments for about 50% of protein pairs with minimal computational effort.
Ego Development and the Ethics of Care and Justice: The Relations Among Them Revisited
Authors: Skoe Eva E. A. , von der Lippe Anna L.
Published: July 2002

ABSTRACT This study examined the links among ego development and the ethics of care and justice in 144 Norwegian men and women, 15 to 48 years old, taking into consideration age, sex, education, and verbal intelligence. As expected, the relationship between Loevinger's model of ego development and care‐based moral reasoning as measured with Skoe's Ethic of Care Interview (ECI) was significantly stronger than the one between ego development and justice as measured with Rest's Defining Issues Test (DIT). Both ethics correlated significantly with verbal ability. Analyses showed that beyond its overlap with verbal intelligence, the variance shared between the ECI and ego development was substantial. By contrast, when verbal intelligence was controlled, the DIT was not significantly related to ego development or to the care ethic.
The Effect of Maternal Support for Counting and Cardinal Understanding in Pre‐School Children
Authors: Linnell Maggie , Fluck Michael
Published: July 2002

The effect of maternal support on the development of counting and cardinality was examined by observing 18 mother‐child dyads engaged in two procedurally similar , but conceptually different tasks in a longitudinal design consisting of three phases (32, 38 and 44 months). At 32 and 38 months of age in both the supported and a similar unsupported condition the children were more successful in the count task than in the give ‘x’task, which requires knowledge of the relationship between counting and cardinality. Observations of mother‐child interactions found that whilst maternal support for the count word sequence was similar in the two tasks the way mothers focused their child’s attention on the objects in the set differed between the tasks. It is argued that social biases as well as cognitive ones constrain the development of cardinal understanding.
Towards a unified framework for high and low frequency return volatility modeling
Authors: Andersen T. G. , Bollerslev T.
Published: July 2002

This paper provides a selective summary of recent work that has documented the usefulness of high‐frequency, intraday return series in exploring issues related to the more commonly studied daily or lower‐frequency returns. We show that careful modeling of intraday data helps resolve puzzles and shed light on controversies in the extant volatility literature that are difficult to address with daily data. Among other things, we provide evidence on the interaction between market microstructure features in the data and the prevalence of strong volatility persistence, the source of significant day‐of‐the‐week effect in daily returns, the apparent poor forecast performance of daily volatility models, and the origin of long‐memory characteristics in daily return volatility series.
Abnormal returns, risk, and options in large data sets
Authors: Caserta S. , DanÃÂÃÂelsson J. , De Vries C. G.
Published: July 2002

Large data sets in finance with millions of observations have become widely available. Such data sets enable the construction of reliable semi‐parametric estimates of the risk associated with extreme price movements. Our approach is based on semi‐parametric statistical extreme value analysis, and compares favorably with the conventional finance normal distribution based approach. It is shown that the efficiency of the estimator of the extreme returns may benefit from high frequency data. Empirical tail shapes are calculated for the German Mark—US Dollar foreign exchange rate, and we use the semi‐parametric tail estimates in combination with the empirical distribution function to evaluate the returns on exotic options.
Resonant Wave Interaction with Random Forcing and Dissipation
Authors: Milewski Paul A. , Tabak Esteban G. , Vanden‐Eijnden Eric
Published: July 2002

A new model for studying energy transfer is introduced. It consists of a “resonant duo”—a resonant quartet where extra symmetries support a reduced subsystem with only two degrees of freedom—where one mode is forced by white noise and the other is damped. This system has a single free parameter: the quotient of the damping coefficient to the amplitude of the forcing times the square root of the strength of the nonlinearity. As this parameter varies, a transition takes place from a Gaussian, high‐temperature, near equilibrium regime, to one highly intermittent and non‐Gaussian. Both regimes can be understood in terms of appropriate Fokker–Planck equations.
Spatial characteristics of sarcoplasmic reticulum Ca2+ release events triggered by L‐type Ca2+ current and Na+ current in guinea‐pig cardiac myocytes
Authors: Lipp Peter , Egger Marcel , Niggli Ernst
Published: July 2002

Ca2+ signals in cardiac muscle cells are composed of spatially limited elementary events termed Ca2+ sparks. Several studies have also indicated that Ca2+ signals smaller than Ca2+ sparks can be elicited. These signals have been termed Ca2+ quarks and were proposed to result from the opening of a single Ca2+ release channel of the sarcoplasmic reticulum. We used laser‐scanning confocal microscopy to examine the subcellular properties of Na+ current (INa)‐ and L‐type Ca2+ current (ICa,L)‐induced Ca2+ transients in voltage‐clamped ventricular myocytes isolated from guinea‐pigs. Both currents, INa and ICa,L, evoked substantial, global Ca2+ transients. To examine the spatiotemporal properties of such Ca2+ signals, we performed power spectral analysis of these Ca2+ transients and found that both lacked spatial frequency components characteristic for Ca2+ sparks. The application of 10 μm verapamil to partially block L‐type Ca2+ current reduced the corresponding Ca2+ transients down to individual Ca2+ sparks. In contrast, INa‐induced Ca2+ responses were still spatially homogeneous and lacked Ca2+ sparks even for small current amplitudes. By using high resistance patch pipettes (> 4 MΩ) to exaggerate the loss of voltage control during INa, Ca2+ sparks appeared superimposed on a homogeneous Ca2+ release component and were exclusively triggered during the flow of INa. In the presence of 10 μm ryanodine both ICa,L and INa elicited small, residual Ca2+ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that INa is indeed able to cause Ca2+ release in guinea‐pig ventricular myocytes. In contrast to ICa,L‐induced Ca2+ transients, which are built up from the recruitment of individual Ca2+ sparks, the INa‐evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca2+ release event is distinct from the Ca2+ spark. Thus, INa‐induced Ca2+ transients are composed of smaller Ca2+ signals, most likely Ca2+ quarks.
Oxidative capacity interacts with oxygen delivery to determine maximal O2 uptake in rat skeletal muscles in situ
Authors: Hepple Russell T. , Hagen Jason L. , Krause Dan J.
Published: July 2002

Based on proportional changes in V̇O2,max with alterations in O2 delivery, it is widely held that O2 availability limits V̇O2,max. In contrast, reductions in V̇O2,max are also seen when mitochondrial oxidative capacity is reduced. Taken collectively, these prior results are consistent with the notion that there is not a single‐step limitation to V̇O2,max. We used a pump‐perfused rat hindlimb model to test the hypothesis that combining moderate reductions in O2 delivery and mitochondrial oxidative capacity would yield a greater reduction in V̇O2,max than seen when performing each intervention independently, demonstrating an interaction between O2 supply and mitochondrial oxidative capacity in determining V̇O2,max. Four groups of animals were studied: two in high O2 delivery conditions (hindlimb O2 delivery: 88 ± 1 μmol O2 min−1; mean ±s.e.m.) and two in moderately reduced O2 delivery conditions (66 ± 2 μmol O2 min−1). One group at each level of O2 delivery was treated with 0.1 μM myxothiazol to reduce mitochondrial oxidative capacity via competitive inhibition of NADH cytochrome c reductase. V̇O2,max in control animals (no myxothiazol) was 29 % lower in the moderately reduced O2 delivery group (592 ± 24 mmol O2 min−1 (100 g)−1); P < 0.05) than in the high O2 delivery group (833 ± 63 μmol O2 min−1 (100 g)−1). Similarly, V̇O2,max was reduced by 29 % (594 ± 22 μmol O2 min−1 (100 g)−1); P < 0.05) in myxothiazol‐treated animals in high O2 delivery conditions compared to control animals in high O2 delivery conditions. When myxothiazol treatment was combined with moderately reduced O2 delivery, V̇O2,max was reduced by an additional 18 % (484 ± 21 μmol O2 min−1 (100 g)−1); P < 0.05) compared to either intervention performed independently. These results show that O2 supply and mitochondrial oxidative capacity interact to determine V̇O2,max.
Mobility of ions, sugar, and water in the cytoplasm of Xenopus oocytes expressing Na+‐coupled sugar transporters (SGLT1)
Authors: Zeuthen Thomas , Zeuthen Emil , Klaerke Dan A.
Published: July 2002

A model was set up to study water transport in membrane proteins expressed in Xenopus oocytes. The model was tested experimentally using human and rabbit Na+‐glucose cotransporters (SGLT1), and was used to explain controversies regarding unstirred layer effects. Cotransport of Na+, sugar and water was monitored by two‐electrode voltage clamp and online measurements of oocyte volume. The specific resistance of the oocyte cytoplasm was found by means of microelectrodes to be 263 ± 91 Ω cm (s.d., n= 52), or 2.5 times that of Kulori medium, in agreement with reported values of intracellular ion concentrations and diffusion constants. Osmotically induced volume and resistance changes were compatible with a model of the oocyte in which 37 ± 17 % (s.d., n= 66) of the intracellular volume acts as a free solution while the remainder is inert, being occupied by organelles, etc. The model explains the results of several types of experiments: rapid changes in rates of water cotransport induced by changes in clamp voltage followed by osmotic equilibration in sugar‐free conditions; volume changes induced by Na+ transport via the ionophore gramicidin; and uphill water transport. Ethanol (0.5 %) induced a marked swelling of the oocytes of about 16 pl s−1. If the specific inhibitor of SGLT1 phlorizin is added from stock solutions in ethanol, the effect of ethanol obfuscates the effects of the inhibitor. We conclude that the transport parameters derived for water cotransport by the SGLT1 can be attributed to the protein residing in the plasma membrane with no significant influences from unstirred layer effects.
The ovine fetal endocrine reflex responses to haemorrhage are not mediated by cardiac nerves
Authors: Wood Charles E.
Published: July 2002

This study was designed to test the hypothesis that cardiac receptors tonically inhibit the secretion of renin, arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) in late‐gestation fetal sheep. Eight chronically catheterised fetal sheep between 122 and 134 days gestation were subjected to injection or infusion of saline or 4 % procaine into the pericardial space. Fetal blood pressure and heart rate were monitored and fetal blood samples were drawn to measure the response to these injections. Injection of procaine into the pericardial space effectively blocked cardiac nerves, as evidenced by a reduction in the variability of fetal heart rate and by the blockade of reflex reductions in fetal heart rate after intravenous injection of phenylephrine (an α‐adrenergic agonist which raises blood pressure). Injection of saline had no discernable effects on any of the measured variables. A single injection of procaine, followed by a slow infusion, produced a transient blockade of cardiac nerves. Multiple injections of procaine produced a sustained blockade of cardiac nerves and a sustained rise in fetal plasma renin activity and ACTH. In none of the experiments did procaine significantly alter fetal plasma AVP concentrations. In 11 fetuses between 121 and 134 days gestation, we combined the cardiac nerve blockade with slow haemorrhage to test the cardiac nerves as mediators of the endocrine response to haemorrhage in utero. Cardiac nerve blockade exaggerated the fetal blood gas response to haemorrhage somewhat but did not significantly alter the magnitude of the ACTH, AVP, or plasma renin activity response to haemorrhage. We conclude that cardiac nerves in the late‐gestation fetal sheep have minor influences on plasma renin activity and ACTH in normovolaemic fetuses, but that changes in cardiac nerve activity do not mediate the endocrine responsiveness to haemorrhage.
Topographic reorganization in area 18 of adult cats following circumscribed monocular retinal lesions in adolescence
Authors: Young J. M. , Waleszczyk W. J. , Burke W. , Calford M. B. , Dreher B.
Published: July 2002

Circumscribed laser lesions were made in the nasal retinae of one eye in adolescent cats. Ten to sixteen months later, about 80 % of single neurones recorded in the lesion projection zone (LPZ) of contralateral area 18 (parastriate cortex, area V2) were binocular but when stimulated via the lesioned eye had ectopic discharge fields (displaced to normal retina in the vicinity of the lesion). Although the clear majority of binocular cells recorded from the LPZ responded with higher peak discharge rates to stimuli presented via the non‐lesioned eye, the orientation and direction selectivities as well as preferred and upper cut‐off velocities for stimuli presented through either eye were very similar. Furthermore, the sizes of the ectopic discharge fields of binocular cells recorded from the LPZ were not significantly different from those of their counterparts plotted via the non‐lesioned eye. Thus, monocular retinal lesions performed in adolescent cats induce topographic reorganization in the LPZ of area 18. Although a similar reorganization occurs in area 17 (striate cortex, area V1) of cats in which monocular retinal lesions were made either in adulthood or adolescence, in view of the very different velocity response profiles of ectopic discharge fields in areas 17 and those in area 18, it appears that ectopic discharge fields in area 17 are largely independent of excitatory feedback input from area 18.
Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord
Authors: Jang Il‐Sung , Jeong Hyo‐Jin , Katsurabayashi Shutaro , Akaike Norio
Published: July 2002

GABAA receptor‐mediated presynaptic depolarization is believed to induce presynaptic inhibition of excitatory synaptic transmission. We report here the functional roles of presynaptic GABAA receptors in glycinergic transmission of the rat spinal cord. In mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with native glycinergic and GABAergic nerve terminals, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated from a mixture of both glycinergic and GABAergic sIPSCs by perfusing the SDCN nerve cell body with ATP‐free internal solution. Under such experimental conditions, exogenously applied muscimol (0.5 μM) depolarized glycinergic presynaptic nerve terminals and significantly increased glycinergic sIPSC frequency to 542.7 ± 47.3 % of the control without affecting the mean current amplitude. The facilitatory effect of muscimol on sIPSC frequency was completely blocked by bicuculline (10 μM) or SR95531 (10 μM), selective GABAA receptor antagonists. This muscimol‐induced presynaptic depolarization was due to a higher intraterminal Cl− concentration, which is maintained by a bumetanide‐sensitive Na‐K‐Cl cotransporter. On the contrary, when electrically evoked, this muscimol‐induced presynaptic depolarization was found to decrease the action potential‐dependent glycine release evoked by focal stimulation of a single terminal. The results suggest that GABAA receptor‐mediated presynaptic depolarization has two functional roles: (1) presynaptic inhibition of action potential‐driven glycinergic transmission, and (2) presynaptic facilitation of spontaneous glycinergic transmission.
Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons
Authors: Glazebrook Patricia A. , Ramirez Angelina N. , Schild John H. , Shieh Char‐Chang , Doan Thanh , Wible Barbara A. , Kunze Diana L.
Published: July 2002

Voltage‐gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0‐3) and in 85‐90 % of the neurons from older animals (postnatal days 21‐60). In voltage clamp studies, α‐dendrotoxin (α‐DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K+ currents of the sensory neurons. α‐DTX blocked current in both A‐ and C‐type neurons. The current had characteristics of a delayed rectifier with activation positive to −50 mV and little inactivation during 250 ms pulses. In current‐clamp experiments α‐DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A‐ and C‐type neurons. However, there were prominent effects on excitability. α‐DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after‐hyperpolarization and increased the frequency/pattern of discharge of A‐ and C‐type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K+ currents function in response to the loss of the α‐DTX‐sensitive current to effect these changes in action potential wave shape and discharge.
No evidence of an intracellular lactate shuttle in rat skeletal muscle
Authors: Sahlin Kent , Fernström Maria , Svensson Michael , Tonkonogi Michail
Published: July 2002

The concerted view is that cytosolic pyruvate is transferred into mitochondria and after oxidative decarboxylation further metabolized in the tricarboxylic acid cycle. Recently this view has been challenged. Based on experimental evidence from rat skeletal muscle it has been concluded that mitochondria predominantly oxidize lactate in vivo and that this constitutes part of an ‘intracellular lactate shuttle’. This view appears to be gaining acceptance in the scientific community and due to its conceptual importance, confirmation by independent experiments is required. We have repeated the experiments in mitochondria isolated from rat soleus muscle. Contrary to the previously published findings we cannot find any mitochondrial respiration with lactate. Analysis of lactate dehydrogenase (LDH) by spectrophotometry demonstrated that the activity in the mitochondrial fraction was only 0.7 % of total activity. However, even when external LDH was added to mitochondria, there were no signs of respiration with lactate. In the presence of conditions where lactate is converted to pyruvate (external additions of both LDH and NAD+), mitochondrial oxygen consumption increased. Furthermore, we provide theoretical evidence that direct mitochondrial lactate oxidation is energetically unlikely. Based on the present data we conclude that direct mitochondrial lactate oxidation does not occur in skeletal muscle. The presence of an ‘intracellular lactate shuttle’ can therefore be questioned.
Hypoxia potentiates exocytosis and Ca2+ channels in PC12 cells via increased amyloid β peptide formation and reactive oxygen species generation
Authors: Green Kim N. , Boyle John P. , Peers Chris
Published: July 2002

Exposure of PC12 cells to chronic hypoxia (CH; 10 % O2, 24 h) augments catecholamine secretion via formation of a Cd2+‐resistant Ca2+ influx pathway, and up‐regulates native L‐type Ca2+ channels. These effects are mimicked by exposure of cells to Alzheimer's disease‐associated amyloid β peptides (AβPs). Since pathological effects of AβPs have been associated with increased levels of reactive oxygen species (ROS), the involvement of ROS in hypoxia‐mediated up‐regulation of exocytosis and Ca2+ channel activity was examined. Both melatonin and ascorbic acid (two structurally unrelated antioxidants) fully blocked the enhancement of catecholamine secretion caused by CH (as determined amperometrically). Enhanced immunofluorescence, observed in chronically hypoxic cells using a primary monoclonal antibody raised against the N‐terminus of AβP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen (an additional antioxidant) also fully prevented augmentation of whole‐cell Ca2+ currents caused by CH (as monitored using whole‐cell patch‐clamp recordings). Exposure of normoxic cells to H2O2 (40 μM, 24 h), like hypoxia, caused Ca2+ channel up‐regulation. Importantly, AβP formation appeared to be an absolute requirement for the effects of hypoxia, since the ability of CH to augment exocytosis and Ca2+ channel activity was blocked by two novel inhibitors of γ secretase, an enzyme complex required for AβP formation. Our results indicate that the effects of hypoxia require ROS generation from AβPs, and suggest that elevated levels of ROS mediate hypoxic and AβP‐mediated pathological remodelling of Ca2+ homeostasis.
Characterization of four types of background potassium channels in rat cerebellar granule neurons
Authors: Han Jaehee , Truell Jeffrey , Gnatenco Carmen , Kim Donghee
Published: July 2002

Cerebellar granule neurons express a standing outward (background) K+ current (IK,SO) that regulates the resting membrane potential and cell excitability. As several tandem‐pore (2P) K+ channel mRNAs are highly expressed in cerebellar granule cells, we studied whether, and which, 2P K+ channels contribute to IK,SO. IK,SO was highly sensitive to changes in extracellular pH and was partially inhibited by acetylcholine, as reported previously. In cell‐attached patches from cultured cerebellar granule neurons, four types of K+ channels were found to be active when membrane potential was held at −50 mV or +50 mV in symmetrical 140 mm) KCl. Based on single‐channel conductances, gating kinetics and modulation by pharmacological agents and pH, three K+ channels could be considered as functional correlates of TASK‐1, TASK‐3 and TREK‐2, which are members of the 2P K+ channel family. The fourth K+ channel (Type 4) has not been described previously and its molecular correlate is not yet known. Based on the measurement of channel current densities, the Type 2 (TASK‐3) and the Type 4 K+ channels were determined to be the major sources of IK,SO in cultured cerebellar granule neurons. The Type 1 (TASK‐1) and Type 3 (TREK‐2) activities were relatively low throughout cell growth in culture (1‐10 days). Similar to TASK‐1 and TASK‐3, the Type 4 K+ channel was highly sensitive to changes in extracellular pH, showing a 78 % inhibition by changing the extracellular pH from 7.3 to 6.3. The results of this study show that three 2P K+ channels and an additional pH‐sensing K+ channel (Type 4) comprise the IK,SO in cultured cerebellar granule neurons. Our results also show that the high sensitivity of IK,SO to extracellular pH comes from the high sensitivity of Type 2 (TASK‐3) and Type 4 K+ channels.
Convergent and reciprocal modulation of a leak K+ current and Ih by an inhalational anaesthetic and neurotransmitters in rat brainstem motoneurones
Authors: Sirois Jay E. , Lynch Carl , Bayliss Douglas A.
Published: July 2002

Neurotransmitters and volatile anaesthetics have opposing effects on motoneuronal excitability which appear to reflect contrasting modulation of two types of subthreshold currents. Neurotransmitters increase motoneuronal excitability by inhibiting TWIK‐related acid‐sensitive K+ channels (TASK) and shifting activation of a hyperpolarization‐activated cationic current (Ih) to more depolarized potentials; on the other hand, anaesthetics decrease excitability by activating a TASK‐like current and inducing a hyperpolarizing shift in Ih activation. Here, we used whole‐cell recording from motoneurones in brainstem slices to test if neurotransmitters (serotonin (5‐HT) and noradrenaline (NA)) and an anaesthetic (halothane) indeed compete for modulation of the same ion channels ‐ and we determined which prevails. When applied together under current clamp conditions, 5‐HT reversed anaesthetic‐induced membrane hyperpolarization and increased motoneuronal excitability. Under voltage clamp conditions, 5‐HT and NA overcame most, but not all, of the halothane‐induced current. When Ih was blocked with ZD 7288, the neurotransmitters completely inhibited the K+ current activated by halothane; the halothane‐sensitive neurotransmitter current reversed at the equilibrium potential for potassium (EK) and displayed properties expected of acid‐sensitive, open‐rectifier TASK channels. To characterize modulation of Ih in relative isolation, effects of 5‐HT and halothane were examined in acidified bath solutions that blocked TASK channels. Under these conditions, 5‐HT and halothane each caused their characteristic shift in voltage‐dependent gating of Ih. When tested concurrently, however, halothane decreased the neurotransmitter‐induced depolarizing shift in Ih activation. Thus, halothane and neurotransmitters converge on TASK and Ih channels with opposite effects; transmitter action prevailed over anaesthetic effects on TASK channels, but not over effects on Ih. These data suggest that anaesthetic actions resulting from effects on either TASK or hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels in motoneurones, and perhaps at other CNS sites, can be modulated by prevailing neurotransmitter tone.
Pacemaker shift in the gastric antrum of guinea‐pigs produced by excitatory vagal stimulation involves intramuscular interstitial cells
Authors: Hirst G. D. S. , Dickens E. J. , Edwards F. R.
Published: July 2002

Intracellular recordings were made from isolated bundles of the circular muscle layer of guinea‐pig gastric antrum and the responses produced by stimulating intrinsic nerve fibres were examined. After abolishing the effects of stimulating inhibitory nerve terminals with apamin and l‐nitroarginine (NOLA), transmural nerve stimulation often evoked a small amplitude excitatory junction potential (EJP) and invariably evoked a regenerative potential. Neurally evoked regenerative potentials had similar properties to those evoked in the same bundle by direct stimulation. EJPs and neurally evoked regenerative potentials were abolished by hyoscine suggesting that both resulted from the release of acetylcholine and activation of muscarinic receptors. Neurally evoked regenerative potentials, but not EJPs, were abolished by membrane hyperpolarization, caffeine and chloride channel blockers. In the intact antrum, excitatory vagal nerve stimulation increased the frequency of slow waves. Simultaneous intracellular recordings of pacemaker potentials from myenteric interstitial cells (ICCMY) and slow waves showed that the onset of each pacemaker potential normally preceded the onset of each slow wave but vagal stimulation caused the onset of each slow wave to precede each pacemaker potential. Together the observations suggest that during vagal stimulation there is a change in the origin of pacemaker activity with slow waves being initiated by intramuscular interstitial cells (ICCIM) rather than by ICCMY.
Stimulation‐dependent regulation of the ph, volume and quantal size of bovine and rodent secretory vesicles
Authors: Pothos Emmanuel N. , Mosharov Eugene , Liu Kuo‐Peing , Setlik Wanda , Haburcak Marian , Baldini Giulia , Gershon Michael D. , Tamir Hadassah , Sulzer David
Published: July 2002

Trapping of weak bases was utilized to evaluate stimulus‐induced changes in the internal pH of the secretory vesicles of chromaffin cells and enteric neurons. The internal acidity of chromaffin vesicles was increased by the nicotinic agonist 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP; in vivo and in vitro) and by high K+ (in vitro); and in enteric nerve terminals by exposure to veratridine or a plasmalemmal [Ca2+]o receptor agonist (Gd3+). Stimulation‐induced acidification of chromaffin vesicles was [Ca2+]o‐dependent and blocked by agents that inhibit the vacuolar proton pump (vH+‐ATPase) or flux through Cl− channels. Stimulation also increased the average volume of chromaffin vesicles and the proportion that displayed a clear halo around their dense cores (called active vesicles). Stimulation‐induced increases in internal acidity and size were greatest in active vesicles. Stimulation of chromaffin cells in the presence of a plasma membrane marker revealed that membrane was internalized in endosomes but not in chromaffin vesicles. The stable expression of botulinum toxin E to prevent exocytosis did not affect the stimulation‐induced acidification of the secretory vesicles of mouse neuroblastoma Neuro2A cells. Stimulation‐induced acidification thus occurs independently of exocytosis. The quantal size of secreted catecholamines, measured by amperometry in cultured chromaffin cells, was found to be increased either by prior exposure to L‐DOPA or stimulation by high K+, and decreased by inhibition of vH+‐ATPase or flux through Cl− channels. These observations are consistent with the hypothesis that the content of releasable small molecules in secretory vesicles is increased when the driving force for their uptake is enhanced, either by increasing the transmembrane concentration or pH gradients.
Salmon calcitonin – a potent inhibitor of food intake in states of impaired leptin signalling in laboratory rodents
Authors: Eiden Sandra , Daniel Carolin , Steinbrueck Alexandra , Schmidt Ingrid , Simon Eckhart
Published: July 2002

To compare the anorectic effectiveness of leptin and the amylin analogue salmon calcitonin (sCT), rodents were treated on 1 day with subcutaneous injections. In chow‐fed C57Bl/6J mice, leptin and sCT reduced energy intake and acted additively. After C57Bl/6J mice had become leptin‐resistant on being fed chocolate as a palatable high‐caloric supplement to chow, their sCT‐induced decrease in energy intake was more pronounced than in chow‐fed mice with differential changes in the intake of chocolate (strong reduction) and chow (slight increase). Dose‐response relationships for sCT‐induced reductions in energy intake were analysed in chow‐fed C57Bl/6J mice and two obese strains, ob/ob mice and melanocortin‐4 receptor knockout (MC4‐r‐KO) mice, as well as in wild‐type and fatty (fa/fa) rats. Compared to C57Bl/6J mice, reduction in food intake induced by sCT was attenuated in MC4‐r‐KO mice, and nearly absent in ob/ob mice, over the dose range investigated. Compared to C57Bl/6J mice, wild‐type rats responded more sensitively to sCT and its efficiency was only slightly reduced in fatty (fa/fa) rats. Thus, while genetically induced failures of leptin signalling reduce the action of sCT, it effectively inhibits the intake of a palatable, high fat‐high sugar diet even in states of diet‐induced obesity with functional leptin resistance.
Modulation of volume‐sensitive chloride current by noradrenaline in rabbit portal vein myocytes
Authors: Ellershaw D. C. , Greenwood I. A. , Large W. A.
Published: July 2002

The effect of noradrenaline on the volume‐sensitive chloride current (ICl(swell)) was studied with conventional whole‐cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of ICl(swell) in some cells and a decrease in others. In the presence of the β‐adrenoceptor antagonist propranolol, noradrenaline increased ICl(swell) and in the presence of the α1‐adrenoceptor antagonist prazosin, noradrenaline reduced ICl(swell). The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of ICl(swell) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol‐12‐myristate‐13‐acetate (PMA) and phorbol‐12,13‐dibutyrate (PDBu) increased the amplitude of ICl(swell) by approximately 60 and 100 %, respectively, in a voltage‐independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin‐C decreased the amplitude of ICl(swell) in a concentration‐dependent but voltage‐independent manner. Bath application of 8‐Br‐cAMP decreased ICl(swell) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of ICl(swell) by approximately 80–90 %. In the presence of propranolol, chelerythrine prevented the increase of ICl(swell) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances ICl(swell) by acting on α1‐adrenoceptors and reduces ICl(swell) by stimulating β‐adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.
Influence of hypernatraemia and urea excretion on the ability to excrete a maximally hypertonic urine in the rat
Authors: Cheema‐Dhadli Surinder , Halperin Mitchell L.
Published: July 2002

Rats normally excrete 20‐25 mmol of sodium (Na+) + potassium (K+) per kilogram per day. To minimize the need for a large water intake, they must excrete urine with a very high electrolyte concentration (tonicity). Our objective was to evaluate two potential factors that could influence the maximum urine tonicity, hypernatraemia and the rate of urea excretion. Balance studies were carried out in vasopressin‐treated rats fed a low‐electrolyte diet. In the first series, the drinking solution contained an equivalent sodium chloride (NaCl) load at 150 or 600 mmol l−1. In the second series, the maximum urine tonicity was evaluated in rats consuming 600 mmol l−1 NaCl with an 8‐fold range of urea excretion. Hypernatraemia (148 ± 1 mmol l−1) developed in all rats that drank 600 mmol l−1 saline. Although the rate of Na++ K+ excretion was similar in both saline groups, the maximum urine total cation concentration was significantly higher in the hypernatraemic group (731 ± 31 vs. 412 ± 37 mmol l−1). Only when the rate of excretion of urea was very low, was there a further increase in the maximum urine total cation concentration (1099 ± 118 mmol l−1). Thus hypernatraemia was the most important factor associated with a higher urine tonicity.
β‐Adrenergic stimulation modulates Ca2+‐ and voltage‐dependent inactivation of L‐type Ca2+ channel currents in guinea‐pig ventricular myocytes
Authors: Findlay Ian
Published: July 2002

The objective of this study was to examine the effect of β‐adrenergic stimulation upon voltage‐ and Ca2+‐induced inactivation of native cardiac L‐type Ca2+ channels. Whole‐cell currents were recorded from guinea‐pig isolated ventricular myocytes. Total and voltage‐dependent inactivation was separated by replacing extracellular Ca2+ with Mg2+. L‐type Ca2+ channel behaviour was monitored with outward Ca2+ channel currents. First, the voltage dependence of inactivation was studied at fixed times (50 and 1000 ms) after activation. This showed that under control conditions Ca2+ contributed little to inactivation. In isoproterenol (isoprenaline), voltage‐dependent inactivation was markedly reduced and Ca2+ contributed largely to total inactivation. Second, the time dependence of inactivation was studied at a fixed voltage (+10 mV). In control conditions the fast phase of inactivation (τf≈15 ms) was reduced to the same extent by ryanodine (τf≈30 ms) and the absence of Ca2+ (τf≈30 ms) while the slow phase of inactivation (τs≈70 ms) was reduced by ryanodine (τs≈160 ms) and further reduced in the absence of Ca2+ (τs≈300 ms). In isoproterenol, biphasic inactivation of Ca2+ currents (τf≈4 ms, τs≈60 ms) was replaced by a single slow (τ≈450 ms) phase of inactivation in the absence of Ca2+. It is concluded that, under control conditions Ca2+ channel current decay is largely dominated by rapid voltage‐dependent inactivation, while in isoproterenol this is replaced by Ca2+‐induced inactivation.
Dynamic Modeling of a Cogenerating Nuclear Gas Turbine Plant—Part I: Modeling and Validation
Authors: Kikstra J. F. , Verkooijen A. H. M.
Published: July 2002

The high-temperature gas-cooled reactor is a promising concept for inherently safe nuclear power generation. This article deals with dynamic modeling of a combined heat and power plant, based on a helium-cooled reactor in combination with a closed-cycle gas turbine system. A one-dimensional flow model describing the helium flow and the two-phase water flow is used through the whole plant, with different source terms in different pieces of equipment. A stage-by-stage model is produced for the radial compressor and axial turbine. Other models include the recuperator, water/helium heat exchangers, a natural convection evaporator, valves, etc. In Part II the model will be used to analyze the dynamic behavior and to design a control system.
Dynamic Modeling of a Cogenerating Nuclear Gas Turbine Plant—Part II: Dynamic Behavior and Control
Authors: Kikstra J. F. , Verkooijen A. H. M.
Published: July 2002

Using the dynamic model of the cogenerating nuclear gas turbine plant developed in Part I of this article, the dynamic behavior of this plant is analyzed and a control structure is designed. First it is determined how several design choices affect the system dynamics. Then the requirements and options for a control system design are investigated. A number of possible control valve positions in the flowsheet are tested with transients in order to make an argued choice. The model is subsequently used to determine the optimal working conditions for different heat and power demands, these are used as set-points for the control system. Then the interaction between manipulated and controlled variables is mapped and based on this information a choice for coupling them in decentralized feedback control loops is made. This control structure is then tuned and tested. It can be concluded that both heat and power demand can be followed with acceptable performance over a wide range.
Dynamic Behavior of a Thin-Walled Cylindrical Workpiece During the Turning-Cutting Process, Part 2: Experimental Approach and Validation
Authors: Mehdi K. , Rigal J.-F. , Play D.
Published: July 2002

Chatter vibrations in the cutting process have a central place in many machining applications. A numerical and theoretical approach of self-excited vibrations during the turning process of thin-walled hollow workpieces has been presented in the accompanying paper. Furthermore, a finite element model has been proposed to simulate the dynamics of the system. The response to a Dirac excitation, presented as Nyquist curves, is proposed in order to characterize the dynamics of the turning process and the stability criterion. In this the second part of two related papers, the main objective is to validate the simulated dynamic behavior by using the experimental approach. The results of machining tests performed on thin-walled tubes with steel and aluminum alloys, using different operating conditions (dimensions, geometry and setting conditions) are presented and discussed.