bluuebunny/crossref_metadata_2025 huggingface dataset:
Visual evaluationin vivoof ‘complexion radiance’ using the C.L.B.T.™ sensory methodology
Authors: Musnier C. , Piquemal P. , Beau P. , Pittet J. C.
Published: January 2004

Purpose:The aim of this study was to develop a new technique enabling visual evaluationin vivoof ‘complexion radiance’ based on sensory analysis, and to apply this methodology to a practical case.

Methods:This evaluation, conducted by trained assessors, was based on a visual analysisin vivoof the following seven descriptors, clearly defined by consensus: a red‐pink, olive, beige, and light‐pink skin coloring (C), as well as the luminosity (L), brightness (B), and transparency (T) of each subject's facial skin. The scoring of perceived skin descriptor intensity was made possible with the help of structured and analogical scales. Once the assessors were considered to be accurate and repeatable, a method was designed to evaluate a cosmetic product's effect on complexion radiance. The study was conducted on 20 healthy female subjects, between 20 and 35 years of age, who met the ‘dull complexion’ criteria. Product ‘A’ (Oligo 25, Laboratoires Vichy, Asnieres, France), formulated to improve ‘complexion radiance,’ was applied by each subject for 28 days, once a day in the morning, in a homogeneous fashion over the entire face, using a standard quantity and a specific way of massaging the product in. The clinical parameters were evaluated at T0, T+1 h, and at T+28 days. This test was a non‐comparative open study and each subject served as her own reference.

Results:One hour after its application, product A produced an immediate significant effect by decreasing the complexion's ‘olive’ skin cast and improving its ‘light‐pink’ coloring, its ‘luminosity,’ and ‘brightness.’ The data concerning the long‐term effect of product A following 28 days of application showed that there was significant improvement in these first four descriptors, while the ‘beige’ skin coloring was ultimately diminished in addition.

Conclusion:Thisin vivoC.L.B.T.™ sensory methodology is a technique enabling the visual evaluation of a product's multi‐factorial claim that it improves ‘the complexion's radiance.’ It made it possible to show, in a reliable and repeatable fashion, the efficacy of a cosmetic product in improving the ‘radiance of the complexion’ of members of a test panel. This methodology also substantiated the immediate and long‐term effects produced by product A, which made the skin pinker and less sallow, more luminous, more uniform (skin coloring), more regular (skin texture), and then lighter. To conclude, five descriptors out of the seven determining complexion radiance were improved.
Ultrasonographic subepidermal low‐echogenic band, dependence of age and body site
Authors: Sandby‐møller Jane , Wulf Hans Christian
Published: January 2004

Background/purpose: The presence of an age‐dependent subepidermal low‐echogenic band (SLEB) is well established at specific body sites. Several studies have also proposed it to be a marker of photoaging, but only few studies have included children in their material, and UVR exposure has predominantly been evaluated as differences between body sites with different UVR exposure. The aim of this study was to further investigate the existence of SLEB and exploring its relation to both age and body site/UVR exposure.

Methods: In all 137 healthy volunteers from 4 to 68 years of age were included. In vivo high‐frequency ultrasonography (B‐mode, 20 MHz) was obtained from sun‐exposed and sun‐protected skin in a period with negligible environmental exposure to solar UVR. The SLEB was evaluated both visually and quantitatively.

Results: We found a significant correlation between visual scoring of SLEB and dermal echogenicity, which supports the usefulness of SLEB scoring.

We found that the grade (existence and development) of SLEB increased significantly with age at both the forehead and at the dorsal aspect of the forearm, where a well‐defined SLEB was absent in children and present in over 50% of adults above the age of 40 years. No such age dependence was found either at the buttock or at the shoulder, and remarkably a well‐defined SLEB was present in 37% of the adolescents at the buttock.

Conclusion: These results indicate that the age‐related increase in SLEB grade at highly sun‐exposed body sites might be attributed to UVR exposure. The presence of SLEB in both the young and at the sun‐protected buttock points to a multifactorial etiology, where anatomical differences may play an important role as well as water content. SLEB might not be a very sensitive and specific marker of photoaging.
Dynamic properties of corticogeniculate excitatory transmission in the rat dorsal lateral geniculate nucleusin vitro
Authors: Granseth Björn
Published: January 2004

The feedback excitation from the primary visual cortex to principal cells in the dorsal lateral geniculate nucleus (dLGN) is markedly enhanced with firing frequency. This property presumably reflects the ample short‐term plasticity at the corticogeniculate synapse. The present study aims to explore corticogeniculate excitatory postsynaptic currents (EPSCs) evoked by brief trains of stimulation with whole‐cell patch‐clamp recordings in dLGN slices from DA‐HAN rats. The EPSCs rapidly increased in amplitude with the first two or three impulses followed by a more gradual growth. A double exponential function with time constants 39 and 450 ms empirically described the growth for 5–25Hz trains. For lower train frequencies (down to 1Hz) a third component with time constant 4.8 s had to be included. The different time constants are suggested to represent fast and slow components of facilitation and augmentation. The time constant of the fast component changed with the extracellular calcium ion concentration as expected for a facilitation mechanism involving an endogenous calcium buffer that is more efficiently saturated with larger calcium influx. Concerning the function of the corticogeniculate feedback pathway, the different components of short‐term plasticity interacted to increase EPSC amplitudes on a linear scale to firing frequency in the physiological range. This property makes the corticogeniculate synapse well suited to function as a neuronal amplifier that enhances the thalamic transfer of visual information to the cortex.
Sub‐ and suprathreshold receptive field properties of pyramidal neurones in layers 5A and 5B of rat somatosensory barrel cortex
Authors: Manns Ian D. , Sakmann Bert , Brecht Michael
Published: January 2004

Layer 5 (L5) pyramidal neurones constitute a major sub‐ and intracortical output of the somatosensory cortex. This layer 5 is segregated into layers 5A and 5B which receive and distribute relatively independent afferent and efferent pathways. We performed in vivo whole‐cell recordings from L5 neurones of the somatosensory (barrel) cortex of urethane‐anaesthetized rats (aged 27–31 days). By delivering 6 deg single whisker deflections, whisker pad receptive fields were mapped for 16 L5A and 11 L5B neurones located below the layer 4 whisker‐barrels. Average resting membrane potentials were −75.6 ± 1.1 mV, and spontaneous action potential (AP) rates were 0.54 ± 0.14 APs s−1. Principal whisker (PW) evoked responses were similar in L5A and L5B neurones, with an average 5.0 ± 0.6 mV postsynaptic potential (PSP) and 0.12 ± 0.03 APs per stimulus. The layer 5A sub‐ and suprathreshold receptive fields (RFs) were more confined to the principle whisker than those of layer 5B. The basal dendritic arbors of layer 5A and 5B cells were located below both layer 4 barrels and septa, and the cell bodies were biased towards the barrel walls. Responses in both L5A and L5B developed slowly, with onset latencies of 10.1 ± 0.5 ms and peak latencies of 33.9 ± 3.3 ms. Contralateral multi‐whisker stimulation evoked PSPs similar in amplitude to those of PW deflections; whereas, ipsilateral stimulation evoked smaller and longer latency PSPs. We conclude that in L5 a whisker deflection is represented in two ways: focally by L5A pyramids and more diffusely by L5B pyramids as a result of combining different inputs from lemniscal and paralemniscal pathways. The relevant output evoked by a whisker deflection could be the ensemble activity in the anatomically defined cortical modules associated with a single or a few barrel‐columns.
Augmentation of corticogeniculate EPSCs in principal cells of the dorsal lateral geniculate nucleus of the rat investigated in vitro
Authors: Granseth Björn , Lindström Sivert
Published: January 2004

Augmentation is a component of short‐term synaptic plasticity with a gradual onset and duration in seconds. To investigate this component at the corticogeniculate synapse, whole cell patch‐clamp recordings were obtained from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Trains with 10 stimuli at 25 Hz evoked excitatory postsynaptic currents (EPSCs) that grew in amplitude, primarily from facilitation. Such trains also induced augmentation that decayed exponentially with a time constant τ= 4.6 ± 2.6 s (mean ± standard deviation). When the trains were repeated at 1–10 s intervals, augmentation markedly increased the size of the first EPSCs, leaving late EPSCs unaffected. The magnitude of augmentation was dependent on the number of pulses, pulse rate and intervals between trains. Augmented EPSCs changed proportionally to basal EPSC amplitudes following alterations in extracellular calcium ion concentration. The results indicate that augmentation is determined by residual calcium remaining in the presynaptic terminal after repetitive spikes, competing with fast facilitation. We propose that augmentation serves to maintain a high synaptic strength in the corticogeniculate positive feedback system during attentive visual exploration.
Two modes of polyamine block regulating the cardiac inward rectifier K+ current IK1 as revealed by a study of the Kir2.1 channel expressed in a human cell line
Authors: Ishihara Keiko , Ehara Tsuguhisa
Published: January 2004

The strong inward rectifier K+ current, IK1, shows significant outward current amplitude in the voltage range near the reversal potential and thereby causes rapid repolarization at the final phase of cardiac action potentials. However, the mechanism that generates the outward IK1 is not well understood. We recorded currents from the inside‐out patches of HEK 293T cells that express the strong inward rectifier K+ channel Kir2.1 and studied the blockage of the currents caused by cytoplasmic polyamines, namely, spermine and spermidine. The outward current–voltage (I–V) relationships of Kir2.1, obtained with 5–10μm spermine or 10–100μm spermidine, were similar to the steady‐state outward I–V relationship of IK1, showing a peak at a level that is ∼20mV more positive than the reversal potential, with a negative slope at more positive voltages. The relationships exhibited a plateau or a double‐hump shape with 1μm spermine/spermidine or 0.1μm spermine, respectively. In the chord conductance–voltage relationships, there were extra conductances in the positive voltage range, which could not be described by the Boltzmann relations fitting the major part of the relationships. The extra conductances, which generated most of the outward currents in the presence of 5–10μm spermine or 10–100μm spermidine, were quantitatively explained by a model that considered two populations of Kir2.1 channels, which were blocked by polyamines in either a high‐affinity mode (Mode 1 channel) or a low‐affinity mode (Mode 2 channel). Analysis of the inward tail currents following test pulses indicated that the relief from the spermine block of Kir2.1 consisted of an exponential component and a virtually instantaneous component. The fractions of the two components nearly agreed with the fractions of the blockages in Mode 1 and Mode 2 calculated by the model. The estimated proportion of Mode 1 channels to total channels was 0.9 with 0.1–10μm spermine, 0.75 with 1–100μm spermidine, and between 0.75 and 0.9 when spermine and spermidine coexisted. An interaction of spermine/spermidine with the channel at an intracellular site appeared to modify the equilibrium of the two conformational channel states that allow different modes of blockage. Our results suggest that the outward IK1 is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward IK1, not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block.
Effects of cytosolic NADH/NAD+ levels on sarcoplasmic reticulum Ca2+ release in permeabilized rat ventricular myocytes
Authors: Zima Aleksey V. , Copello Julio A. , Blatter Lothar A.
Published: January 2004

In the heart ischaemic conditions induce metabolic changes known to have profound effects on Ca2+ signalling during excitation–contraction coupling. Ischaemia also affects the redox state of the cell. However, the role of cytosolic redox couples, such as the NADH/NAD+ redox system, for the regulation of Ca2+ homeostasis has remained elusive. We studied the effects of NADH and NAD+ on sarcoplasmic reticulum (SR) Ca2+ release in permeabilized rat ventricular myocytes as well as on Ca2+ uptake by SR microsomes and ryanodine receptor (RyR) single channel activity. Exposure of permeabilized myocytes to NADH (2 mm; [Ca2+]cyt= 100nm) decreased the frequency and the amplitude of spontaneous Ca2+ sparks by 62% and 24%, respectively. This inhibitory effect was reversed by NAD+ (2 mm) and did not depend on mitochondrial function. The inhibition of Ca2+ sparks by NADH was associated with a 52% decrease in SR Ca2+ load. Some of the effects observed with NADH may involve the generation of superoxide anion (O2−·) as they were attenuated to just a transient decrease of Ca2+ spark frequency by superoxide dismutase (SOD). O2−· generated in situ from the xanthine/xanthine oxidase reaction caused a slowly developing decrease of Ca2+ spark frequency and SR Ca2+ load by 44% and 32%, respectively. Furthermore, in studies with cardiac SR microsomes NADH slowed the rate of ATP‐dependent Ca2+ uptake by 39%. This effect also appeared to depend on O2−· formation. Single channel recordings from RyRs incorporated into lipid bilayers revealed that NADH (2 mm) inhibited the activity of RyR channels by 84%. However, NADH inhibition of RyR activity was O2−·‐independent. In summary, an increase of the cytoplasmic NADH/NAD+ ratio depresses SR Ca2+ release in ventricular cardiomyocytes. The effect appears to be mediated by direct NADH inhibition of RyR channel activity and by indirect NADH inhibition (O2−· mediated) of SR Ca2+‐ATPase activity with a subsequent decrease in SR Ca2+ content.
Creatine supplementation increases glucose oxidation and AMPK phosphorylation and reduces lactate production in L6 rat skeletal muscle cells
Authors: Ceddia Rolando B. , Sweeney Gary
Published: January 2004

Recent observations have suggested that creatine supplementation might have a beneficial effect on glucoregulation in skeletal muscle. However, conclusive studies on the direct effects of creatine on glucose uptake and metabolism are lacking. The objective of this study was to investigate the effects of creatine supplementation on basal and insulin‐stimulated glucose transporter (GLUT4) translocation, glucose uptake, glycogen content, glycogen synthesis, lactate production, glucose oxidation and AMP‐activated protein kinase (AMPK) phosphorylation in L6 rat skeletal muscle cells. Four treatment groups were studied: control, insulin (100 nm), creatine (0.5 mm) and creatine + insulin. After 48 h of creatine supplementation the creatine and phosphocreatine contents of L6 myoblasts increased by ∼9.3‐ and ∼5.1‐fold, respectively, but the ATP content of the cells was not affected. Insulin significantly increased 2‐deoxyglucose uptake (∼1.9‐fold), GLUT4 translocation (∼1.8‐fold), the incorporation of D‐[U‐14C]glucose into glycogen (∼2.3‐fold), lactate production (∼1.5‐fold) and 14CO2 production (∼1.5‐fold). Creatine neither altered the glycogen and GLUT4 contents of the cells nor the insulin‐stimulated rates of 2‐DG uptake, GLUT4 translocation, glycogen synthesis and glucose oxidation. However, creatine significantly reduced by ∼42% the basal rate of lactate production and increased by ∼40% the basal rate of 14CO2 production. This is in agreement with the ∼35% increase in citrate synthase activity and also with the ∼2‐fold increase in the phosphorylation of both α‐1 and α‐2 isoforms of AMPK after creatine supplementation. We conclude that 48 h of creatine supplementation does not alter insulin‐stimulated glucose uptake and glucose metabolism; however, it activates AMPK, shifts basal glucose metabolism towards oxidation and reduces lactate production in L6 rat skeletal muscle cells.
Effect of gastric distension on cardiovascular parameters: gastrovascular reflex is attenuated in the elderly
Authors: Orshoven N. P. van , Oey P. L. , Schelven L. J. van , Roelofs J. M. M. , Jansen P. A. F. , Akkermans L. M. A.
Published: January 2004

Stretching the stomach wall in young healthy subjects causes an increase in muscle sympathetic nerve activity and in blood pressure, the gastrovascular reflex. We compared healthy elderly subjects with healthy young subjects to find out whether the gastrovascular reflex attenuates in normal ageing and we studied whether there was a difference in autonomic function or gastric compliance that could explain this possible attenuation. Muscle sympathetic nerve activity, finger blood pressure and heart rate were continuously recorded during stepwise isobaric gastric distension using a barostat in eight healthy young (6 men and 2 women, 27 ± 3.2 years, mean ±s.e.m.) and eight healthy elderly subjects (7 men and 1 woman, 76 ± 1.5 years). Changes in cardiac output and total peripheral arterial resistance were calculated from the blood pressure signal. The baseline mean arterial pressure and muscle sympathetic nerve activity were higher in the elderly group (both P < 0.05) and muscle sympathetic nerve activity increase during the cold pressor test was lower in the elderly group (P = 0.005). During stepwise gastric distension, the elderly subjects showed an attenuated increase in muscle sympathetic nerve activity compared to the young subjects (P < 0.01). The older group tended to show a higher increase in mean arterial pressure (P = 0.08), heart rate (P = 0.06) and total peripheral arterial resistance (P = 0.09) The cardiac output rose slightly in both groups without significant difference between groups. The fundic compliance did not differ between groups. We conclude that stepwise gastric distension caused an increase in muscle sympathetic nerve activity in both groups, but the increase in the elderly was attenuated.
Developmental regulation of the 5‐HT7 serotonin receptor and transcription factor NGFI‐A in the fetal guinea‐pig limbic system: influence of GCs
Authors: Andrews Marcus H. , Kostaki Alice , Setiawan Elaine , McCabe Lucy , Owen Dawn , Banjanin Sonja , Matthews Stephen G.
Published: January 2004

Fetal exposure to excess glucocorticoids (GCs) programs the developing hypothalamo–pituitary–adrenal (HPA) axis, and may predispose offspring to adult‐onset disease. During development, serotonin (5‐HT) influences transcription of hippocampal GR mRNA via the 5‐HT7 receptor. The effect of 5‐HT on GR involves the transcription factor NGFI‐A. Given the developmental changes which we have previously reported in hippocampal GR mRNA expression, we hypothesized that (1) there are progressive developmental changes in 5‐HT7 receptor and NGFI‐A mRNA expression in the fetal guinea‐pig limbic system, and (2) repeated exposure to synthetic GC treatment will significantly modify developmental expression of these genes. 5‐HT7 receptor mRNA was highly expressed in the hippocampus and thalamus at gestational day (gd) 40 (term ∼70 days), and significantly decreased (P < 0.05) with advancing gestation. Conversely, NGFI‐A mRNA expression in the hippocampus and frontal cortex was almost undetectable at gd40, but was dramatically elevated (P < 0.05; 8‐fold) near term. Changes in mRNA were refelected by NGFI‐A protein levels. These changes were significantly correlated to hippocampal GR expression and fetal plasma cortisol concentrations. Synthetic GC treatment increased NGFI‐A mRNA levels in CA1 and the cingulate cortex, but had no effect on 5‐HT7 receptor expression. In conclusion our results suggest that (1) limbic 5‐HT7 receptor expression is not directly linked to maturation of hippocampal GR in late gestation; (2) the up‐regulation of NGFI‐A expression near term is driven by glucocorticoid; and (3) premature exposure to synthetic glucocorticoid significantly increases NGFI‐A‐related transcriptional activity in the fetal limbic system.
Differential cytokine response in interstitial fluid in skin and serum during experimental inflammation in rats
Authors: Nedrebø Torbjørn , Reed Rolf K. , Jonsson Roland , Berg Ansgar , Wiig Helge
Published: January 2004

Tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) are important mediators produced during inflammation. We hypothesized that the pro‐inflammatory cytokine response in the interstitial fluid (IF) is different from that in serum, and we aimed at quantifying the amount of TNF‐α and IL‐1β in the IF. By centrifugation of rat skin at < 424 g pure IF is extracted. Using ELISA such fluid was analysed for cytokines in back and/or paw skin of pentobarbital‐anaesthetized rats, after either induction of endotoxaemia or ischaemia–reperfusion (I/R) injury. During endotoxaemia, TNF‐α increased in the IF from 0 in control to 640 ± 100 pg ml−1 (mean ±s.e.m.) after 90 min, with the serum concentration being 5–10 times higher at all time points. The response pattern of IL‐1β after lipopolysaccharide (LPS) challenge differed greatly from that of TNF‐α with a large increase in IF from 390 ± 90 to 28 000 ± 1500 pg ml−1 after 210 min, and a significantly smaller increase in serum (600 ± 45 pg ml−1). During reperfusion of the hind paw after 2 h of ischaemia, there was a gradual increase of TNF‐α in both IF of the paw skin and serum after 3 min of reperfusion. Both declined after 20 min. The pattern for IL‐1β differed, increasing significantly less in serum (25 ± 15 pg ml−1 after 20 min of reperfusion) than in the IF (1100 ± 200 pg ml−1). Immunostaining of the inflamed tissues showed increased expression of the two cytokines in cells of both epidermis and dermis compared to controls. Subdermal injections of TNF‐α and IL‐1β at the same concentrations found in IF after LPS infusion affected interstitial fluid pressure significantly. Local TNF‐α production dominates after I/R injury, whereas in endotoxaemia systemic production predominates. For IL‐1β local production dominates in both conditions. Thus, there is a differential pattern of cytokine production and the current method allows the study of the role of cytokines in IF during different inflammatory reactions.
Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control
Authors: Lancaster Matthew K. , Jones Sandra A. , Harrison Simon M. , Boyett Mark R.
Published: January 2004

Recent studies have proposed that release of calcium from the sarcoplasmic reticulum (SR) modulates the spontaneous activity of the sinoatrial node (SAN). Previously we have shown that several calcium regulatory proteins are expressed at a lower level in the centre of the SAN compared with the periphery. Such differences may produce heterogeneity of intracellular calcium handling and pacemaker activity across the SAN. Selective isolations showed that the centre of the SAN is composed of smaller cells than the periphery. Measurements of cytosolic calcium in spontaneously beating cells showed that diastolic calcium, systolic calcium, the calcium transient amplitude and spontaneous rate were greater in larger (likely to be peripheral) cells compared with smaller (likely to be central) SAN cells. The SR calcium content was greater in larger cells, although SR recruitment was more efficient in smaller cells. The sodium–calcium exchanger and sarcolemmal calcium ATPase had a lower activity and the exchanger was responsible for a larger proportion of sarcolemmal calcium extrusion in smaller cells compared with larger cells. Ryanodine had a greater effect on the spontaneous calcium transient in larger cells compared with smaller cells, and slowed pacemaker activity in larger cells but not smaller cells, thus abolishing the difference in cycle length. This study shows heterogeneity of intracellular calcium regulation within the SAN and this contributes to differences in pacemaker activity between cells from across the SAN. The smallest central cells of the leading pacemaker region of the SAN do not require SR calcium for spontaneous activity nor does disruption of the SR alter pacemaking in these primary pacemaker cells.
Modulation of excitatory synaptic transmission in the spinal substantia gelatinosa of mice deficient in the kainate receptor GluR5 and/or GluR6 subunit
Authors: Youn and Dong‐ho , Randić Mirjana
Published: January 2004

Functional kainate (KA) receptors (KARs) are expressed in the spinal cord substantia gelatinosa (SG) region, and their activation has a capacity to modulate excitatory synaptic transmission at primary afferent synapses with SG neurones. In the present study, we have used gene‐targeted mice lacking KAR GluR5 and/or GluR6 subunits to determine the identity of the receptor subunits involved in the KA‐induced modulation of excitatory transmission. Our findings reveal that KARs comprising GluR5 or GluR6 subunits can either suppress or facilitate glutamatergic excitatory transmission in the SG of acutely prepared adult mouse spinal cord slices. In the absence of synaptic inhibition mediated by GABAA and glycine receptors, a biphasic effect of kainate is characteristic with facilitation apparent at a low concentration (30 nm) and depression at a higher concentration (3 μm). In addition, GluR6‐KARs, localizing pre‐ and postsynaptically, are critically involved in inhibiting transmission at both Aδ and C fibre monosynaptic pathways, whereas presynaptic GluR5‐KARs play a limited role in inhibiting the C fibre‐activated pathway. The results obtained support the hypothesis that KARs are involved in bi‐directional regulation of excitatory synaptic transmission in the spinal cord SG region, and that these actions may be of critical importance for nociception and the clinical treatment of pain.
Single cGMP‐activated Ca2+‐dependent Cl‐ channels in rat mesenteric artery smooth muscle cells
Authors: Piper A. S. , Large W. A.
Published: January 2004

The present study describes the single channel properties of a novel cGMP‐activated Ca2+‐dependent Cl− channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell‐attached patches in the presence of 8Br cGMP in response to the addition of caffeine or noradrenaline and in both outside‐out and inside‐out patches when the internal patch surface was bathed in cGMP and Ca2+. The channels were permeable to Cl− ions with an anion permeability sequence of SCN− (1.7) > Cl− (1.0) > I− (0.6). Single channel mean open probability (NPo) was independent of voltage and the channels displayed three conductance levels of 15, 35 and 55 pS. cGMP was required for channel activation and the single channel NPo increased sharply with raised [Ca2+]i, maximal activation occurring at a [Ca2+]i of about 100 nm. The relationship between NPo and cGMP concentration was voltage independent and could be fitted by the Hill equation giving a Kd of about 3 μm and a Hill coefficient (nH) of 3. cGMP‐ and Ca2+‐dependent channel currents were inhibited by 10 μm ZnCl2 but niflumic acid, an inhibitor of Ca2+‐activated Cl− channels, had no effect. Inhibition of cGMP‐dependent protein kinase activity by the cGMP‐dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP‐PNP reduced NPo, while activation of cGMP‐dependent protein kinase by guanosine 3′, 5′‐cyclic monophosphate, β‐phenyl‐1, N2‐etheno‐8‐bromo‐sodium salt (8Br PET cGMP) produced a significant increase in single channel NPo. It is likely that these single channel currents underlie the noradrenaline‐activated inward current important for vasomotion in these resistance arteries.
Basolateral Na+–H+ exchanger‐1 in rat taste receptor cells is involved in neural adaptation to acidic stimuli
Authors: Lyall Vijay , Alam Rammy I. , Malik Shahbaz A. , Phan Tam‐Hao T. , Vinnikova Anna K. , Heck Gerard L. , DeSimone John A.
Published: January 2004

The role of basolateral Na+–H+ exchanger isoform‐1 (NHE‐1) was investigated in neural adaptation of rat taste responses to acidic stimuli, by direct measurement of intracellular pH (pHi) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. In TRCs perfused with CO2/HCO3−‐free solution (pH 7.4), removal of basolateral Na+ decreased pHi reversibly and zoniporide, a specific NHE‐1 blocker, inhibited the Na+‐induced changes in pHi. The spontaneous rate of TRC pHi recovery from NH4Cl pulses was inhibited by basolateral zoniporide with a Ki of 0.33μm. Exposure to basolateral ionomycin, reversibly increased TRC Ca2+, resting pHi, and the spontaneous rate of pHi recovery from an NH4Cl pulse. These effects of Ca2+ on pHi were blocked by zoniporide. In in vivo experiments, topical lingual application of zoniporide increased the magnitude of the CT responses to acetic acid and CO2, but not to HCl. Topical lingual application of ionomycin did not affect the phasic part of the CT responses to acidic stimuli, but decreased the tonic part by 50% of control over a period of about 1 min. This increased adaptation in the CT response was inhibited by zoniporide. Topical lingual application of 8‐CPT‐cAMP increased the CT responses to HCl, but not to CO2, and acetic acid. In the presence of cAMP, ionomycin increased sensory adaptation to HCl, CO2, and acetic acid. Thus, cAMP and Ca2+ independently modulate CT responses to acidic stimuli. While cAMP enhances TRC apical H+ entry and CT responses to strong acid, an increase in Ca2+ activates NHE‐1, and increases neural adaptation to all acidic stimuli.
A pharmacological profile of the aldehyde receptor repertoire in rat olfactory epithelium
Authors: Araneda Ricardo C. , Peterlin Zita , Zhang Xinmin , Chesler Alex , Firestein Stuart
Published: January 2004

Several lines of evidence suggest that odorants are recognized through a combinatorial process in the olfactory system; a single odorant is recognized by multiple receptors and multiple odorants are recognized by the same receptor. However few details of how this might actually function for any particular odour set or receptor family are available. Approaching the problem from the ligands rather than the receptors, we used the response to a common odorant, octanal, as the basis for defining multiple receptor profiles. Octanal and other aldehydes induce large EOG responses in the rodent olfactory epithelium, suggesting that these compounds activate a large number of odour receptors (ORs). Here, we have determined and compared the pharmacological profile of different octanal receptors using Ca2+ imaging in isolated olfactory sensory neurones (OSNs). It is believed that each OSN expresses only one receptor, thus the response profile of each cell corresponds to the pharmacological profile of one particular receptor. We stimulated the cells with a panel of nine odorants, which included octanal, octanoic acid, octanol and cinnamaldehyde among others (all at 30μm). Cluster analysis revealed several distinct pharmacological profiles for cells that were all sensitive to octanal. Some receptors had a broad molecular range, while others were activated only by octanal. Comparison of the profiles with that of the one identified octanal receptor, OR‐I7, indicated several differences. While OR‐I7 is activated by low concentrations of octanal and blocked by citral, other receptors were less sensitive to octanal and not blocked by citral. A lower estimate for the maximal number of octanal receptors is between 33 and 55. This large number of receptors for octanal suggests that, although the peripheral olfactory system is endowed with high sensitivity, discrimination among different compounds probably requires further central processing.
Effects of strength training on muscle lactate release and MCT1 and MCT4 content in healthy and type 2 diabetic humans
Authors: Juel Carsten , Holten Mads K. , Dela Flemming
Published: January 2004

Lactate is released from skeletal muscle in proportion to glucose uptake rates, and it leaves the cells via simple diffusion and two monocarboxylate transporter proteins, MCT1 and MCT4. In reponse to endurance training MCT1 – and possibly MCT4 – content in muscle increases. The MCTs have not previously been measured in patients with type 2 diabetes (Type 2), and the response to strength training is unknown. Ten Type 2 and seven healthy men (Control) strength‐trained one leg (T) 3 times a week for 6 weeks while the other leg remained untrained (UT). Each session lasted no more than 30 min. After strength training, muscle biopsies were obtained and an isoglycaemic, hyperinsulinaemic clamp, combined with arterial and femoral venous catheterization of both legs, was carried out. During hyperinsulinaemia lactate release was always increased in T versus UT legs. MCT1 was lower (P<0.05) and MCT4 similar in Type 2 versus Control. With training, MCT1 content always increased, while MCT4 only increased in Control. Conclusions: MCT1 content in skeletal muscle in Type 2 is lower compared with healthy men. Strength training increases MCT1 content in healthy men and in Type 2, thus normalizing the content in Type 2.
Roles of compatible osmolytes and heat shock protein 70 in the induction of tolerance to stresses in porcine endothelial cells
Authors: Alfieri Roberta R. , Petronini Pier Giorgio , Bonelli Mara A. , Desenzani Silvia , Cavazzoni Andrea , Borghetti Angelo F. , Wheeler Kenneth P.
Published: January 2004

Studies of the responses of porcine pulmonary endothelial cells to acute hypertonic stress have been extended by examining the induction and underlying mechanisms of cell tolerance to both osmotic and heat stresses. Preliminary adaptation of these cells to 0.4osmol (kg H2O)−1 rendered them tolerant either to subsequent severe osmotic stress (0.7osmol (kg H2O)−1) or to subsequent severe heat shock (50 min at 49°C). In contrast, preliminary exposure of the cells to mild heat shock (44°C for 30 min) induced tolerance only to severe heat shock, not to hyperosmotic stress. Induction of tolerance to heat shock by either procedure correlated with the induced expression of heat shock protein 70 (HSP70). Induction of tolerance to hyperosmotic stress, on the other hand, was associated with the cellular accumulation of osmolytes, such as amino acids, betaine and myo‐inositol, and did not correlate with the induced expression of HSP70. It also required a reduction in the final change of osmotic pressure applied to the cells, such that maximum cell shrinkage would not be much more than 40%. In general, therefore, HSP70 and compatible osmolytes have distinct roles in cellular adaptation to these stresses.
Rat transcobalamin: cloning and regulation of mRNA expression
Authors: Kalra Seema , Seetharam Shakuntla , Yammani Raghunatha R. , Seetharam Bellur
Published: January 2004

Transcobalamin (TC) has been cloned and used for studying its gene expression in the rat. TC mRNA is distributed widely in adult rat tissues, but at different levels (kidney > liver > lung > yolk sac > intestine > heart > brain > spleen > muscle). TC mRNA levels were 4‐fold higher in the jejunum and ileum compared to its levels in the duodenum. During postnatal development, TC mRNA levels in the ileum declined 4‐fold from day 4 to day 12, but increased by 5‐fold between days 12 and 24. In contrast, TC mRNA levels increased by 2.5‐fold in the kidney from day 4 to day 12 and then declined by 2‐fold by day 24. Adrenalectomy of adult rats resulted in a 4‐fold decline in ileal levels of TC mRNA and a 50% decline in the ileal mucosal formation of the TC‐[57Co] cobalamin (Cbl) complex following oral administration of [57Co]Cbl complexed to gastric intrinsic factor (IF). Cortisone treatment reversed these changes noted in the ileum. In contrast to ileum, kidney TC mRNA levels were not altered significantly in adrenalectomized rats before and after cortisone treatment. Taken together, this study has provided evidence for the regulation of TC gene expression in the rat kidney and intestine during their postnatal development, and cortisone selectively regulates ileal but not kidney TC mRNA levels.
Calcium‐induced calcium release regulates action potential generation in guinea‐pig sympathetic neurones
Authors: Locknar Sarah A. , Barstow Karen L. , Tompkins John D. , Merriam Laura A. , Parsons Rodney L.
Published: January 2004

Experiments were done using guinea‐pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium‐induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca2+]i) with the Ca2+‐sensitive dye fluo‐3, we demonstrated that 10 mm caffeine activated ryanodine receptors and caused a rise in [Ca2+]i in both Ca2+‐containing and Ca2+‐deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μm thapsigargin or caffeine and 20 μm ryanodine blocked subsequent caffeine‐induced elevations of [Ca2+]i. Treatment with thapsigargin, ryanodine or 200 μm Cd2+ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μm tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca2+ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea‐pig sympathetic stellate neurones.
Medullary serotonergic neurones and adjacent neurones that express neurokinin‐1 receptors are both involved in chemoreception in vivo
Authors: Nattie Eugene E. , Li Aihua , Richerson George , Lappi Douglas A.
Published: January 2004

Neurokinin‐1 receptor (NK1R)‐expressing neurones that are involved in chemoreception at the retrotrapezoid nucleus (Nattie & Li, 2002b) are also prominent at locations that contain medullary serotonergic neurones, which are chemosensitive in vitro. In medullary regions containing both types, we evaluated their role in central chemoreception by specific cell killing. We injected (2×100 nl) (a) substance P–saporin (SP‐SAP; 1μm) to kill NK1R‐expressing neurones, (b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti‐SERT‐SAP; 1μm) to kill serotonergic neurones, or (c) SP‐SAP and anti‐SERT‐SAP together to kill both types. Controls received IgG‐SAP injections (1μm). There was no double‐labelling of NK1R‐immunoreactive (ir) and tryptophan‐hydroxylase (TPOH)‐ir neurones. Cell (somatic profile) counts showed that NK1R‐ir neurones in the SP‐SAP group were reduced by 31%; TPOH‐ir neurones in the anti‐SERT‐SAP group by 28%; and NK1R‐ir and TPOH‐ir neurones, respectively, in the combined lesion group by 55% and 31% (P < 0.001; two‐way ANOVA; P < 0.05, Tukey's post hoc test). The treatments had no significant effect on sleep/wake time, body temperature, or oxygen consumption but all three reduced the ventilatory response to 7% inspired CO2 in wakefulness and sleep by a similar amount. SP‐SAP treatment decreased the averaged CO2 responses (3, 7 and 14 days after lesions) in wakefulness and sleep by 21% and 16%, anti‐SERT‐SAP decreased the responses by 15% and 18%, and the combined treatment decreased the responses by 12% and 12% (P < 0.001; two‐way ANOVA; P < 0.05, Tukey's post hoc test). We conclude that separate populations of serotonergic and adjacent NK1R‐expressing neurones in the medulla are both involved in central chemoreception in vivo.
The voltage‐dependent ClC‐2 chloride channel has a dual gating mechanism
Authors: Zúñiga Leandro , Niemeyer María Isabel , Varela Diego , Catalán Marcelo , Cid L. Pablo , Sepúlveda Francisco V.
Published: January 2004

Functional and structural studies demonstrate that Cl− channels of the ClC family have a dimeric double‐barrelled structure, with each monomer contributing an identical pore. Single protopore gating is a fast process dependent on Cl− interaction within the selectivity filter and in ClC‐0 has a low temperature coefficient over a 10°C range (Q10). A slow gating process closes both protopores simultaneously, has a high Q10, is facilitated by extracellular Zn2+ and Cd2+ and is abolished or markedly reduced by mutation of a cysteine conserved in ClC‐0, ‐1 and ‐2. In order to test the hypothesis that similar slow and fast gates exist in the widely expressed ClC‐2 Cl− channel we have investigated the effects of these manoeuvres on ClC‐2. We find that the time constants of both components of the double‐exponential hyperpolarization‐dependent activation (and deactivation) processes have a high temperature dependence, with Q10 values of about 4–5, suggesting important conformational changes of the channel. Mutating C256 (equivalent to C212 in ClC‐0) to A, led to a significant fraction of constitutively open channels at all potentials. Activation time constants were not affected but deactivation was slower and significantly less temperature dependent in the C256A mutant. Extracellular Cd2+, that inhibits wild‐type (WT) channels almost fully, inhibited C256A only by 50%. In the WT, the time constants for opening were not affected by Cd2+ but deactivation at positive potentials was accelerated by Cd2+. This effect was absent in the C256A mutant. The effect of intracellular Cl− on channel activation was unchanged in the C256A mutant. Collectively our results strongly support the hypothesis that ClC‐2 possesses a common gate and that part of the current increase induced by hyperpolarization represents an opening of the common gate. In contrast to the gating in ClC‐0, the protopore gate and the common gate of ClC‐2 do not appear to be independent.
Transient receptor potential‐like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones
Authors: Bengtson C. Peter , Tozzi Alessandro , Bernardi Giorgio , Mercuri Nicola B.
Published: January 2004

Transient receptor potential (TRP) channels form cationic channels activated by diverse factors including mechanical stimuli, changes in osmolarity, pH and temperature, as well as the exogenous irritant, capsaicin. Metabotropic glutamate receptors have also recently been linked to TRP channel activation in neurones of the substantia nigra, hippocampus and cerebellum, suggesting a novel role for such channels in synaptic communication via endogenous neurotransmitters. We tested this for dopamine neurones in rat brain slices by characterizing the current–voltage relationship and pharmacology of EPSCs mediated by group I metabotropic glutamate receptor subtype 1 (mGluR1). Slow inward currents (273 ± 35 pA peak amplitude, 381 ± 25 ms latency, holding potential (Vh) =−73 mV) representing evoked mGluR1 EPSCs were isolated in the presence of antagonists of AMPA, NMDA, GABAA, GABAB, muscarinic and glycine receptors. CPCCOEt (100 μm), an mGluR1 antagonist, blocked the residual EPSC in all recordings. mGluR1‐activated EPSCs reversed polarity near −10 mV, consistent with the involvement of a cationic channel. Extracellular application of the non‐selective TRP channel blockers SKF 96365, flufenamic acid and ruthenium red caused reversible inhibition of mGluR1‐activated EPSCs. These characteristics parallel those of mGluR1 activation with an agonist and indicate the involvement of a TRP‐like channel in mGluR1‐mediated EPSCs.
Depletion of substance P and glutamate by capsaicin blocks respiratory rhythm in neonatal rat in vitro
Authors: Morgado‐Valle Consuelo , Feldman Jack L.
Published: January 2004

The specific role of the neuromodulator substance P (SP) and its target, the neurokinin 1 receptor (NK1R), in the generation and regulation of respiratory activity is not known. The preBötzinger complex (preBötC), an essential site for respiratory rhythm generation, contains glutamatergic NK1R‐expressing neurones that are strongly modulated by exogenously applied SP or acute pharmacological blockade of NK1Rs. We investigated the effects of capsaicin, which depletes neuropeptides (including SP) and glutamate from presynaptic terminals, on respiratory motor output in medullary slice preparations of neonatal rat that generate respiratory‐related activity. Bath application of capsaicin slowed respiratory motor output in a dose‐ and time‐dependent manner. Respiratory rhythm could be restored by bath application of SP or glutamate transporter blockers. Capsaicin also evoked dose‐dependent glutamate release and depleted SP in fibres within the preBötC. Our results suggest that depletion of SP (or other peptides) and/or glutamate by capsaicin causes a cessation of respiratory rhythm in neonatal rat slices.
Differential Representation of Perception and Action in the Frontal Cortex
Authors: Schwartz Andrew B. , Moran Daniel W. , Reina G. Anthony
Published: January 2004

A motor illusion was created to separate human subjects' perception of arm movement from their actual movement during figure drawing. Trajectories constructed from cortical activity recorded in monkeys performing the same task showed that the actual movement was represented in the primary motor cortex, whereas the visualized, presumably perceived, trajectories were found in the ventral premotor cortex. Perception and action representations can be differentially recognized in the brain and may be contained in separate structures.
Computational Constraints on Syntactic Processing in a Nonhuman Primate
Authors: Fitch W. Tecumseh , Hauser Marc D.
Published: January 2004

The capacity to generate a limitless range of meaningful expressions from a finite set of elements differentiates human language from other animal communication systems. Rule systems capable of generating an infinite set of outputs (“grammars”) vary in generative power. The weakest possess only local organizational principles, with regularities limited to neighboring units. We used a familiarization/discrimination paradigm to demonstrate that monkeys can spontaneously master such grammars. However, human language entails more sophisticated grammars, incorporating hierarchical structure. Monkeys tested with the same methods, syllables, and sequence lengths were unable to master a grammar at this higher, “phrase structure grammar” level.
A New Strategy for the Synthesis of Glycoproteins
Authors: Zhang Zhiwen , Gildersleeve Jeff , Yang Yu-Ying , Xu Ran , Loo Joseph A. , Uryu Sean , Wong Chi-Huey , Schultz Peter G.
Published: January 2004

Posttranslational modifications of proteins regulate many biological processes, including metabolism, signal transduction, and gene expression. The synthetic challenges associated with generating homogeneous populations of selectively modified proteins, however, have hindered detailed studies of the effects of these modifications on protein structure and function. Here, we report an approach to the cotranslational synthesis of selectively glycosylated proteins in which the modified amino acid is genetically encoded. We show that myoglobin containing β–
N
-acetylglucosamine (GlcNAc)–serine at a defined position can be expressed in
Escherichia coli
in good yield and with high fidelity. The β-GlcNAc moiety can be recognized by a saccharide-binding protein, or subsequently modified with a galactosyltransferase to build more complex carbohydrates. This approach should be generally applicable to other posttranslational modifications such as protein phosphorylation, acetylation, and methylation.