bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Assays Demonstrate That Pollen Tube Organelles Use Kinesin-Related Motor Proteins to Move along Microtubules[W]
Authors: Romagnoli Silvia , Cai Giampiero , Cresti Mauro
Published: January 2003

The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro–polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.
AtATMIs Essential for Meiosis and the Somatic Response to DNA Damage in Plants[W]
Authors: Garcia Valérie , Bruchet Hugues , Camescasse Delphine , Granier Fabienne , Bouchez David , Tissier Alain
Published: January 2003

In contrast to yeast or mammalian cells, little is known about the signaling responses to DNA damage in plants. We previously characterized AtATM, an Arabidopsis homolog of the human ATM gene, which is mutated in ataxia telangiectasia, a chromosome instability disorder. The Atm protein is a protein kinase whose activity is induced by DNA damage, particularly DNA double-strand breaks. The phosphorylation targets of Atm include proteins involved in DNA repair, cell cycle control, and apoptosis. Here, we describe the isolation and functional characterization of two Arabidopsis mutants carrying a T-DNA insertion in AtATM. Arabidopsis atm mutants are hypersensitive to γ-radiation and methylmethane sulfonate but not to UV-B light. In correlation with the radiation sensitivity, atm mutants failed to induce the transcription of genes involved in the repair and/or detection of DNA breaks upon irradiation. In addition, atm mutants are partially sterile, and we show that this effect is attributable to abundant chromosomal fragmentation during meiosis. Interestingly, the transcription of DNA recombination genes during meiosis was not dependent on AtATM, and meiotic recombination occurred at the same rate as in wild-type plants, raising questions about the function of AtAtm during meiosis in plants. Our results demonstrate that AtATM plays a central role in the response to both stress-induced and developmentally programmed DNA damage.
Three Isoforms of Isoamylase Contribute Different Catalytic Properties for the Debranching of Potato Glucans[W]
Authors: Hussain Hasnain , Mant Alexandra , Seale Robert , Zeeman Sam , Hinchliffe Edward , Edwards Anne , Hylton Christopher , Bornemann Stephen , Smith Alison M. , Martin Cathie , Bustos Regla
Published: January 2003

Isoamylases are debranching enzymes that hydrolyze α-1,6 linkages in α-1,4/α-1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.
Dual Role of TOC1 in the Control of Circadian and Photomorphogenic Responses in Arabidopsis[W]
Authors: Más Paloma , Alabadí David , Yanovsky Marcelo J. , Oyama Tokitaka , Kay Steve A.
Published: January 2003

To examine the role of the TOC1 (TIMING OF CAB EXPRESSION1) gene in the Arabidopsis circadian system, we generated a series of transgenic plants expressing a gradation in TOC1 levels. Silencing of the TOC1 gene causes arrhythmia in constant darkness and in various intensities of red light, whereas in blue light, the clock runs faster in silenced plants than in wild-type plants. Increments in TOC1 gene dosage delayed the pace of the clock, whereas TOC1 overexpression abolished rhythmicity in all light conditions tested. Our results show that TOC1 RNA interference and toc1-2 mutant plants displayed an important reduction in sensitivity to red and far-red light in the control of hypocotyl elongation, whereas increments in TOC1 gene dosage clearly enhanced light sensitivity. Furthermore, the red light–mediated induction of CCA1/LHY expression was decreased in TOC1 RNA interference and toc1-2 mutant plants, indicating a role for TOC1 in the phytochrome regulation of circadian gene expression. We conclude that TOC1 is an important component of the circadian clock in Arabidopsis with a crucial function in the integration of light signals to control circadian and morphogenic responses.
ETHYLENE RESPONSE FACTOR1 Integrates Signals from Ethylene and Jasmonate Pathways in Plant Defense[W]
Authors: Lorenzo Oscar , Piqueras Raquel , Sánchez-Serrano Jose J. , Solano Roberto
Published: January 2003

Cross-talk between ethylene and jasmonate signaling pathways determines the activation of a set of defense responses against pathogens and herbivores. However, the molecular mechanisms that underlie this cross-talk are poorly understood. Here, we show that ethylene and jasmonate pathways converge in the transcriptional activation of ETHYLENE RESPONSE FACTOR1 (ERF1), which encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression. The expression of ERF1 can be activated rapidly by ethylene or jasmonate and can be activated synergistically by both hormones. In addition, both signaling pathways are required simultaneously to activate ERF1, because mutations that block any of them prevent ERF1 induction by any of these hormones either alone or in combination. Furthermore, 35S:ERF1 expression can rescue the defense response defects of coi1 (coronative insensitive1) and ein2 (ethylene insensitive2); therefore, it is a likely downstream component of both ethylene and jasmonate signaling pathways. Transcriptome analysis in Col;35S:ERF1 transgenic plants and ethylene/jasmonate-treated wild-type plants further supports the notion that ERF1 regulates in vivo the expression of a large number of genes responsive to both ethylene and jasmonate. These results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes.
Local calcium gradients during excitation–contraction coupling and alternans in atrial myocytes
Authors: Blatter Lothar A. , Kockskämper Jens , Sheehan Katherine A. , Zima Aleksey V. , Hüser Jörg , Lipsius Stephen L.
Published: January 2003

Subcellular Ca2+ signalling during normal excitation‐contraction (E‐C) coupling and during Ca2+ alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca2+ currents (ICa). Ca2+ alternans, a beat‐to‐beat alternation in the amplitude of the [Ca2+]i transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j‐SR) and non‐junctional (nj‐SR) types, both of which have ryanodine‐receptor calcium release channels. During E‐C coupling, Ca2+ entering through voltage‐gated membrane Ca2+ channels (ICa) triggers Ca2+ release at discrete peripheral j‐SR release sites. The discrete Ca2+ spark‐like increases of [Ca2+]i then fuse into a peripheral ‘ring’ of elevated [Ca2+]i, followed by propagation (via calcium‐induced Ca2+ release, CICR) to the cell centre, resulting in contraction. Interrupting ICa instantaneously terminates j‐SR Ca2+ release, whereas nj‐SR Ca2+ release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca2+ alternans. The spatiotemporal [Ca2+]i pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca2+]i and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca2+ alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca2+ waves develop at their border. In conclusion, the results demonstrate that (1) during normal E‐C coupling the atrial [Ca2+]i transient is the result of the spatiotemporal summation of Ca2+ release from individual release sites of the peripheral j‐SR and the central nj‐SR, activated in a centripetal fashion by CICR via ICa and Ca2+ release from j‐SR, respectively, (2) Ca2+ alternans is caused by subcellular alterations of SR Ca2+ release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca2+ waves resulting from heterogeneities in subcellular Ca2+ alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.
AMPA‐sst2 somatostatin receptor interaction in rat hypothalamus requires activation of nmda and/or metabotropic glutamate receptors and depends on intracellular calcium
Authors: Peineau Stéphane , Potier Brigitte , Petit Florence , Dournaud Pascal , Epelbaum Jacques , Gardette Robert
Published: January 2003

Modulation of glutamatergic transmission by neuropeptides is an essential aspect of neuronal network activity. Activation of the hypothalamic somatostatin sst2 receptor subtype by octreotide decreases AMPA glutamate responses, indicating a central link between a neurohormonal and neuromodulatory peptide and the main hypothalamic fast excitatory neurotransmitter. In mediobasal hypothalamic slices, sst2 activation inhibits the AMPA component of glutamatergic synaptic responses but is ineffective when AMPA currents are pharmacologically isolated. In mediobasal hypothalamic cultures, the decrease of AMPA currents induced by octreotide requires a concomitant activation of sst2 receptors with either NMDA and/or metabotropic glutamate receptors. This modulation depends on changes in intracellular calcium concentration induced by calcium flux through NMDA receptors or calcium release from intracellular stores following metabotropic glutamate receptor activation. These results highlight an unusual regulatory mechanism in which the simultaneous activation of at least three different types of receptor is necessary to allow somatostatin‐induced modulation of fast synaptic glutamatergic transmission in the hypothalamus.
Targeting calcium cycling proteins in heart failure through gene transfer
Authors: del Monte Federica , Hajjar Roger J.
Published: January 2003

Our understanding of cardiac excitation‐contraction coupling has improved significantly over the last 10 years. Furthermore, defects in the various steps of excitation‐contraction coupling that characterize cardiac dysfunction have been identified in human and experimental models of heart failure. The various abnormalities in ionic channels, transporters, kinases and various signalling pathways collectively contribute to the ‘failing phenotype.’ However, deciphering the causative changes continues to be a challenge. An important tool in dissecting the importance of the various changes in heart failure has been the use of cardiac gene transfer. To achieve effective cardiac gene transfer a number of obstacles remain, including appropriate vectors for gene delivery, appropriate delivery systems, and a better understanding of the biology of the disease. In this review, we will examine our current understanding of these various factors. Gene transfer provides not only a potential therapeutic modality but also an approach to identifying and validating molecular targets.
Regulation of junctional and non‐junctional sarcoplasmic reticulum calcium release in excitation‐contraction coupling in cat atrial myocytes
Authors: Sheehan Katherine A. , Blatter Lothar A.
Published: January 2003

We have characterized the dependence on membrane potential (Vm) and calcium current (ICa) of calcium‐induced calcium release (CICR) from the junctional‐SR (j‐SR, in the subsarcolemmal (SS) space) and non‐junctional‐SR (nj‐SR, in the central (CT) region of the cell) of cat atrial myocytes using whole‐cell voltage‐clamp together with spatially resolved laser‐scanning confocal microscopy. Subsarcolemmal and central [Ca2+]i transient amplitudes and ICa had a bell‐shaped dependence on Vm, but [Ca2+]i reached a maximum at more negative Vm (‐10 to 0 mV) than ICa (+10 mV). Termination of ICa after a brief depolarization (2.5 to 22.5 ms) immediately interrupted only the SS [Ca2+]i transient, leaving the development of the CT [Ca2+]i transient unaffected. Block of SR function with 20 μm ryanodine and 2 μm thapsigargin, revealed that > 90 % of the control [Ca2+]i transient amplitude was attributable to active SR Ca2+ release through ryanodine receptors (RyRs). The gain of SR Ca2+ release was highest in the SS space at negative test potentials and was less pronounced in the CT region. Inhibition of Na+‐Ca2+ exchange resulted in prolonged and higher amplitude [Ca2+]i transients, elevated resting [Ca2+]i, accelerated propagation of CICR, decreased extrusion of Ca2+ and an increase in j‐SR Ca2+ load. Increasing the cytosolic Ca2+ buffer capacity by internal perfusion with 1 mm EGTA limited SR Ca2+ release to the SS region, indicating that Ca2+ release from nj‐SR is initiated by diffusion of Ca2+ from the cell periphery and propagating CICR. Junctional‐SR Ca2+ release occurred at discrete sites whose order of activation and amplitude of release varied from beat to beat. In conclusion, during normal excitation‐contraction coupling in cat atrial myocytes, only Ca2+ release from the j‐SR is directly activated by Ca2+ entering via ICa. Elevation of SS [Ca2+]i is required to provide the cytosolic Ca2+ gradient needed to initiate regenerative and propagating CICR from nj‐SR.
Pacemaker frequency is increased by sodium nitroprusside in the guinea pig gastric antrum
Authors: Kito Yoshihiko , Suzuki Hikaru
Published: January 2003

In the guinea pig gastric antrum, the effects of sodium nitroprusside (SNP), an NO donor, on pacemaker potentials were investigated in the presence of nifedipine. The pacemaker potentials consisted of primary and plateau components; SNP (> 1 μm) increased the frequency of occurrence of these pacemaker potentials, while inhibiting the plateau component. 1H‐[1,2,4]‐Oxadiazole [4,3‐a] quinoxalin‐1‐one, an inhibitor of guanylate cyclase, had no effect on the excitatory actions of SNP on the frequency of pacemaker potentials. Other types of NO donor, (±)‐S‐nitroso‐N‐acetylpenicillamine, 3‐morpholino‐sydnonimine and 8‐bromoguanosine 3′5’‐cyclic monophosphate had no excitatory effect on pacemaker activity. Forskolin, an activator of adenylate cyclase, or 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulphonic acid, an inhibitor of the Ca2+‐activated Cl− channel, strongly attenuated the generation of pacemaker potentials, and SNP added in the presence of these chemicals restored the generation of pacemaker potentials. The pacemaker potentials evoked by SNP were abolished in low‐Ca2+ solution or by membrane depolarization with high‐K+ solution. The SNP‐induced generation of pacemaker potentials was not prevented by cyclopiazonic acid, an inhibitor of internal Ca2+‐ATPase, but was limited to a transient burst by iodoacetic acid, an inhibitor of glycolysis, carbonyl cyanide m‐chlorophenyl‐hydrazone, a mitochondrial protonophore, or 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N‘,N‘‐tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. These results suggest that the SNP‐induced increase in the frequency of pacemaker potentials is related to the elevated intracellular Ca2+ concentrations due to release from mitochondria, and these actions may be independent of the activation of guanylate cyclase.
Firing behaviour of squirrel monkey eye movement‐related vestibular nucleus neurons during gaze saccades
Authors: McCrea Robert A. , Gdowski Greg T.
Published: January 2003

The firing behaviour of vestibular nucleus neurons putatively involved in producing the vestibulo‐ocular reflex (VOR) was studied during active and passive head movements in squirrel monkeys. Single unit recordings were obtained from 14 position‐vestibular (PV) neurons, 30 position‐vestibular‐pause (PVP) neurons and 9 eye‐head‐vestibular (EHV) neurons. Neurons were sub‐classified as type I or II based on whether they were excited or inhibited during ipsilateral head rotation. Different classes of cell exhibited distinctive responses during active head movements produced during and after gaze saccades. Type I PV cells were nearly as sensitive to active head movements as they were to passive head movements during saccades. Type II PV neurons were insensitive to active head movements both during and after gaze saccades. PVP and EHV neurons were insensitive to active head movements during saccadic gaze shifts, and exhibited asymmetric sensitivity to active head movements following the gaze shift. PVP neurons were less sensitive to ondirection head movements during the VOR after gaze saccades, while EHV neurons exhibited an enhanced sensitivity to head movements in their on direction. Vestibular signals related to the passive head movement were faithfully encoded by vestibular nucleus neurons. We conclude that central VOR pathway neurons are differentially sensitive to active and passive head movements both during and after gaze saccades due primarily to an input related to head movement motor commands. The convergence of motor and sensory reafferent inputs on VOR pathways provides a mechanism for separate control of eye and head movements during and after saccadic gaze shifts.
Effects of ageing and regular aerobic exercise on endothelial fibrinolytic capacity in humans
Authors: Smith Derek T. , Hoetzer Greta L. , Greiner Jared J. , Stauffer Brian L. , DeSouza Christopher A.
Published: January 2003

The capacity of the vascular endothelium locally to release tissue‐type plasminogen activator (t‐PA) is critical for effective endogenous fibrinolysis. We determined the influence of ageing and regular aerobic exercise on the net release of t‐PA across the human forearm in vivo using both cross‐sectional and intervention approaches. First, we studied 62 healthy men aged 22‐35 or 50‐75 years of age who were either sedentary or endurance exercise‐trained. Net endothelial release rates of t‐PA were calculated as the product of the arteriovenous concentration gradient and forearm plasma flow to intra‐arterial bradykinin and sodium nitroprusside. Second, we studied 10 older (60 ± 2 years) healthy sedentary men before and after a 3 month aerobic exercise intervention. Net endothelial t‐PA release was significantly blunted with age in the sedentary men. At the highest dose of bradykinin the increase in t‐PA antigen release was ≈35 % less (P < 0.05) in the older (from −1.0 ± 0.4 to 37.8 ± 3.8 ng (100 ml tissue)−1 min−1) compared with young (from 0.1 ± 0.6 to 56.6 ± 9.2 ng (100 ml tissue)−1 min−1) men. In contrast, the endurance‐trained men did not demonstrate an age‐related decline in the net release of t‐PA antigen. After the exercise intervention, the capacity of the endothelium to release t‐PA increased ≈55 % (P < 0.05) to levels similar to those of the young adults and older endurance‐trained men. Regulated endothelial t‐PA release declines with age in sedentary men. Regular aerobic exercise may not only prevent, but could also reverse the age‐related loss in endothelial fibrinolytic function.
Effects of oxidation and reduction on contractile function in skeletal muscle fibres of the rat
Authors: Lamb G. D. , Posterino G. S.
Published: January 2003

This study investigated the effects of the oxidants hydrogen peroxide (H2O2) and 2,2′‐dithiodipyridine (DTDP), and reductants, glutathione (GSH) and dithiothreitol (DTT), on the properties of the contractile apparatus of rat fast‐ and slow‐twitch skeletal muscle fibres, in order to assess how oxidation affects muscle function. Skinned muscle fibres were activated in heavily‐buffered Ca2+ solutions. The force‐[Ca2+] relationship before and after various treatments was fitted by a Hill curve described by the maximum Ca2+‐activated force, pCa50 (‐log10[Ca2+] giving half‐maximum force) and nH (the Hill coefficient). Exposing freshly skinned fibres to strong reducing conditions (i.e. 10 mm DTT or 5 mm GSH) had little if any effect on Ca2+ sensitivity (pCa50 or nH). The effect of oxidants H2O2 and DTDP depended on whether the fibre was relaxed (in pCa > 9) or activated during the exposure. In both fast‐ and slow‐twitch fibres a 5 min exposure to 10 mm H2O2 at pCa > 9 had no effect on pCa50, causing only a reduction in nH. In contrast, when fast‐twitch fibres were activated in the presence of 10 mm H2O2 (or 100 μm DTDP) there was a substantial increase in pCa50 (by ≈0.06 and 0.1, respectively), as well as larger decreases in nH than occurred in relaxed fibres, with all effects being reversed by DTT (10 mm, 10 min). In slow‐twitch soleus fibres, the activation‐dependent effect of DTDP was even greater (pCa50 increased by ≈0.35), and it was found that the rate of reversal in DTT was also increased by activation. A separate important phenomenon was that fast‐twitch fibres that had been oxidised with H2O2 or DTDP (while either relaxed or activated) showed a paradoxical increase in Ca2+ sensitivity (≈0.04 and 0.25 increase in pCa50, respectively) when briefly exposed to the endogenous reductant GSH (5 mm, 2 min). This effect was reversed by DTT or longer (> 20 min) exposure to GSH, did not occur in slow‐twitch soleus fibres, and may contribute to post‐tetanic potentiation in fast‐twitch muscle. Maximum force was not affected by any of the above treatments, whereas exposure to a high concentration of DTDP (1 mm) did greatly reduce force production. These findings reveal a number of novel and probably important effects of oxidation on the contractile apparatus in skeletal muscle fibres.
Effects of leptin on cat intestinal motility
Authors: Gaigé Stéphanie , Abysique Anne , Bouvier Michel
Published: January 2003

In a previous study, we established that leptin controls food intake and immune responses by acting on intestinal vagal chemosensitive mechanoreceptors via a functional link with interleukin‐1β (Il‐1β). Since the control of intestinal motility is one of the main roles of the vagal afferent fibres, we investigated the effects of leptin on intestinal electromyographic (EMG) activity which reflects intestinal motility. For this purpose, the effects of locally injected leptin on small intestine spontaneous EMG activity were studied in 23 anaesthetised cats. The EMG activity was recorded using bipolar electrodes implanted in the proximal small intestine. Leptin and Il‐1β (0.1, 1 and 10 μg), administered through the artery irrigating the upper part of the intestine 20 min after cholecystokinin (CCK, 10 μg, i.a.), had significant (P < 0.001) excitatory effects on intestinal EMG activity. The effects of both substances were blocked by the endogenous interleukin‐1β receptor antagonist (Il‐1ra, 250 μg, i.a.), by atropine (250 μg, i.a.) and by vagotomy. In the absence of CCK, leptin and Il‐1β had no effect on intestinal electrical activity. It can therefore be concluded that: (1) leptin is effective only after the previous intervention of CCK, (2) the enhancement of the electrical activity induced by leptin involves Il‐1β receptors and the cholinergic excitatory pathway, (3) the modes whereby the leptin‐induced enhancement of EMG activity occurs strongly suggest that these effects are due to a long‐loop reflex involving intestinal vagal afferent fibres and the parasympathetic nervous system.
Chronic inhibition of endothelial nitric oxide synthase activity in nucleus tractus solitarii enhances baroreceptor reflex in conscious rats
Authors: Waki Hidefumi , Kasparov Sergey , Wong Liang‐Fong , Murphy David , Shimizu Tsuyoshi , Paton Julian F. R.
Published: January 2003

In acute experiments, we demonstrated previously that nitric oxide (NO) donors exogenously applied to the nucleus tractus solitarii (NTS) depressed the baroreceptor cardiac reflex. In this study, we determined a role for endogenous endothelial nitric oxide synthase (eNOS) activity in the NTS for chronically regulating baroreceptor reflex function in conscious rats. A recombinant adenoviral vector directing expression of a truncated form of eNOS was microinjected bilaterally into the NTS to inhibit endogenous eNOS activity. Arterial pressure was monitored continuously using radio‐telemetry in freely moving animals and spontaneous baroreceptor reflex gain (sBRG) determined by a time‐series method. sBRG showed a gradual increase from day 7 to 21 after gene transfer and the value at day 21 (1.68 ± 0.20 ms mmHg−1, n= 6) was significantly higher than that before gene transfer (1.13 ± 0.09 ms mmHg−1, P < 0.001). This value was also significantly higher than that in rats in which enhanced green fluorescent protein (eGFP) was expressed in the NTS (1.04 ± 0.21 ms mmHg−1; n= 6, P < 0.01) and saline‐treated groups (1.12 ± 0.15 ms mmHg−1; n= 4, P < 0.05), which did not change from control levels. In addition, heart rate decreased from 336 ± 6 to 318 ± 8 b.p.m. (P < 0.05) 21 days after gene transfer. This value was also significantly lower than that in control groups (eGFP: 348 ± 9 b.p.m., n= 6, P < 0.01; saline: 347 ± 5 b.p.m., n= 4, P < 0.05). Gene transfer did not affect arterial pressure. These findings suggest that in the conscious rat eNOS is constitutively active within the NTS and is a factor regulating baroreceptor reflex gain and heart rate.
The effect of graded exercise on IL‐6 release and glucose uptake in human skeletal muscle
Authors: Helge Jørn W. , Stallknecht Bente , Pedersen Bente Klarlund , Galbo Henrik , Kiens Bente , Richter Erik A.
Published: January 2003

In this study, the hypothesis that the release of interleukin (IL)‐6 from human muscle is linked to exercise intensity and muscle glucose uptake was investigated. In the overnight fasted state, seven healthy males performed knee extension exercise, kicking with both legs, each at 25 % of maximal power (Wmax) for 45 min (eliciting 23 ± 1 % of pulmonary maximal oxygen uptake, V̇O2,max) and then simultaneously with one leg at 65 % and the other leg at 85 % Wmax for 35 min (40 ± 1 % of pulmonary V̇O2,max). Blood was sampled from a femoral artery and both femoral veins, and blood flow was determined by thermodilution. Thigh plasma flow (0.15 ± 0.01, 1.4 ± 0.2, 2.0 ± 0.1 and 2.3 ± 0.2 l min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) and thigh oxygen uptake (0.02 ± 0.01, 0.27 ± 0.03, 0.48 ± 0.04 and 0.55 ± 0.05 l min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) increased with increasing exercise intensity (P < 0.05). Also, thigh IL‐6 release (0.4 ± 0.1, 1.3 ± 0.5, 1.5 ± 0.6 and 2.5 ± 0.7 ng min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) and thigh glucose uptake (0.05 ± 0.01, 0.3 ± 0.05, 0.75 ± 0.16, 1.07 ± 0.15 mmol min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) increased with increasing exercise intensity (P < 0.05). During the last 35 min of exercise, arterial catecholamine concentrations were higher (P < 0.05) than at rest and during low‐intensity exercise. During exercise, thigh IL‐6 release was positively related to both thigh glucose uptake (P < 0.001) and thigh glucose delivery (P < 0.005), but not to thigh glucose extraction. Thigh IL‐6 release was also positively related to arterial plasma adrenaline concentration. The pre‐exercise muscle glycogen concentration tended to correlate with the arteriovenous IL‐6 concentration difference at rest, and the postexercise glycogen concentration was inversely correlated with IL‐6 release during the final 35 min of exercise. In conclusion, the study indicates that IL‐6 release from human muscle is positively related to exercise intensity, arterial adrenaline concentration and muscle glucose uptake. This supports the hypothesis that IL‐6 may be linked to the regulation of glucose homeostasis during exercise. The observation of a relationship between IL‐6 release and muscle glycogen store both at rest and after exercise suggests that IL‐6 may act as a carbohydrate sensor.
Postnatal development of GABAergic signalling in the rat lateral geniculate nucleus: presynaptic dendritic mechanisms
Authors: Perreault Marie‐Claude , Qin Yi , Heggelund Paul , Zhu J. Julius
Published: January 2003

Diverse forms of GABAergic inhibition are found in the mature brain. To understand how this diversity develops, we studied the changes in morphology of inhibitory interneurons and changes in interneuron‐mediated synaptic transmission in the rat dorsal lateral geniculate nucleus (dLGN). We found a steady expansion of the dendritic tree of interneurons over the first three postnatal weeks. During this period, the area around a thalamocortical cell from which GABAA inhibition could be elicited also expanded. Dendritic branching and burst firing in interneurons evolved more slowly. The distal dendrites of interneurons began to branch extensively after the third week, and at the same time burst firing appeared. The appearance of burst firing and an elaborated dendritic tree were accompanied by a pronounced GABAB inhibition of thalamocortical cells. Thus, GABA inhibition of thalamocortical cells developed from one type of GABAA inhibition (spatially restricted) in the young animal into two distinct types of GABAA inhibition (short‐ and long‐range) and GABAB inhibition in the adult animal. The close temporal relationships between the development of the diverse forms of inhibition and the postnatal changes in morphology of local GABAergic interneurons in the dLGN suggest that postnatal dendritic maturation is an important presynaptic factor for the developmental time course of the various types of feedforward inhibition in thalamus.
Cytochrome P450 2C9 plays an important role in the regulation of exercise‐induced skeletal muscle blood flow and oxygen uptake in humans
Authors: Hillig Thore , Krustrup Peter , Fleming Ingrid , Osada Takuya , Saltin Bengt , Hellsten Ylva
Published: January 2003

Previous studies show that exercise‐induced hyperaemia is unaffected by systemic inhibition of nitric oxide synthase (NOS) and it has been proposed that this may be due to compensation by other vasodilators. We studied the involvement of cytochrome P450 2C9 (CYP 2C9) in the regulation of skeletal muscle blood flow in humans and the interaction between CYP 2C9 and NOS. Seven males performed knee extensor exercise. Blood flow was measured by thermodilution and blood samples were drawn frequently from the femoral artery and vein at rest, during exercise and in recovery. The protocol was repeated three times on the same day. The first and the third protocols were controls, and in the second protocol either the CYP 2C9 inhibitor sulfaphenazole alone, or sulfaphenazole in combination with the NOS inhibitor Nω‐monomethyl‐l‐arginine (l‐NMMA) were infused. Compared with control there was no difference in blood flow at any time with sulfaphenazole infusion (P > 0.05) whereas with infusion of sulfaphenazole and l‐NMMA, blood flow during exercise was 16 ± 4 % lower than in control (9 min: 3.67 ± 0.31 vs. 4.29 ± 0.20 l min−1; P < 0.05). Oxygen uptake during exercise was 12 ± 3 % lower (9 min: 525 ± 46 vs. 594 ± 24 ml min−1; P < 0.05) with co‐infusion of sulfaphenazole and l‐NMMA, whereas oxygen uptake during sulfaphenazole infusion alone was not different from that of control (P > 0.05). The results demonstrate that CYP 2C9 plays an important role in the regulation of hyperaemia and oxygen uptake during exercise. Since inhibition of neither NOS nor CYP 2C9 alone affect skeletal muscle blood flow, an interaction between CYP 2C9 and NOS appears to exist so that a CYP‐dependent vasodilator mechanism takes over when NO production is compromised.
Microinjection of muscimol into raphe pallidus suppresses tachycardia associated with air stress in conscious rats
Authors: Zaretsky Dmitry V. , Zaretskaia Maria V. , Samuels Brian C. , Cluxton Laura K. , DiMicco Joseph A.
Published: January 2003

Sympathetically mediated tachycardia is a characteristic feature of the physiological response to emotional or psychological stress in mammals. Activation of neurons in the region of the dorsomedial hypothalamus appears to play a key role in the integration of this response. Tachycardia evoked by chemical stimulation of the dorsomedial hypothalamus can be suppressed by microinjection of the GABAA receptor agonist and neuronal inhibitor muscimol into the raphe pallidus (RP). Therefore, we tested the hypothesis that neuronal excitation in the RP mediates tachycardia seen in experimental air stress in rats. Microinjection of the GABAA receptor antagonist bicuculline methiodide (BMI) into the RP evoked increases in heart rate. At the same sites, microinjection of muscimol (80 pmol (100 nl)−1) had no effect on heart rate under baseline conditions but virtually abolished air stress‐induced tachycardia, while microinjection of lower doses (10 or 20 pmol) produced transient but clear suppression. Microinjection of muscimol at sites outside the RP had no effect on stress‐induced tachycardia, although modest suppression was apparent after injection at two sites within 500 μm of the RP. In another series of experiments, microinjection of muscimol (80 pmol (100 nl)−1) into the RP failed to influence the changes in heart rate produced by baroreceptor loading or unloading. These findings indicate that activity of neurons in the RP plays a previously unrecognized role in the generation of stress‐induced tachycardia.
Heart failure – a challenge to our current concepts of excitation–contraction coupling
Authors: Sjaastad Ivar , Wasserstrom J. Andrew , Sejersted Ole M.
Published: January 2003

Development of novel therapeutic strategies for congestive heart failure (CHF) seems to be hampered by insufficient knowledge of the molecular machinery of excitation‐contraction (EC) coupling in both normal and failing hearts. Cardiac hypertrophy and failure represent a multitude of cardiac phenotypes, and available invasive and non‐invasive techniques, briefly reviewed here, allow proper quantification of myocardial function in experimental models even in rats and mice. Both reduced fractional shortening and reduced velocity of contraction characterize myocardial failure. Only when myocardial function is depressed in vivo can meaningful studies be done in vitro of contractility and EC coupling. Also, we point out potential limitations with the whole cell patch clamp technique. Two main factors stand out as explanations for myocardial failure. First, a basic feature of CHF seems to be a reduced Ca2+ load of the sarcoplasmic reticulum (SR) mainly due to a low phosphorylation level of phospholamban. Second, there seems to be a defect of the trigger mechanism of Ca2+ release from the SR. We argue that this defect only becomes manifest in the presence of reduced Ca2+ reuptake capacity of the SR and that it may not be solely attributable to reduced gain of the Ca2+‐induced Ca2+ release (CICR). We list several possible explanations for this defect that represent important avenues for future research.
Microbial Biodiversity: Approaches to Experimental Design and Hypothesis Testing in Primary Scientific Literature from 1975 to 1999
Authors: Morris Cindy E. , Bardin Marc , Berge Odile , Frey-Klett Pascale , Fromin Nathalie , Girardin Hélène , Guinebretière Marie-Hélène , Lebaron Philippe , Thiéry Jean M. , Troussellier Marc
Published: January 2003

Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.
Role ofRAD52Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair
Authors: Symington Lorraine S.
Published: January 2003

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
Regulation of Bacterial Drug Export Systems
Authors: Grkovic Steve , Brown Melissa H. , Skurray Ronald A.
Published: January 2003

The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.
Biochemistry and Comparative Genomics of SxxK Superfamily Acyltransferases Offer a Clue to the Mycobacterial Paradox: Presence of Penicillin-Susceptible Target Proteins versus Lack of Efficiency of Penicillin as Therapeutic Agent
Authors: Goffin Colette , Ghuysen Jean-Marie
Published: January 2003

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-α and α/β folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK
d
,
d
-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4→3) type. They are inactivated by penicillin and other β-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4→3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze
d
,
d
peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine β-lactamases, and the penicillin sensors of several penicillin sensory transducers help the
d
,
d
-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen
r
protein fusions. Plausible hypotheses are put forward on the roles that the Pen
r
protein fusions, acting as
l
,
d
-acyltransferases, may play in the (3→3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4→3) type to the (3→3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.
A rational approach to uninvestigated dyspepsia in primary care: review of the literature
Authors: Arents N L A , Thijs J C , Kleibeuker J H
Published: January 2003

In this paper the rationale and limitations of the four most important approach strategies to dyspepsia in primary care (empiric treatment, prompt endoscopy, “test-and-scope”, and “test-and-treat”) are analysed. It is concluded that in the absence of alarm symptoms, a “test-and-treat” approach is currently the most rational approach provided that three conditions are met: (1) a highly accurate test should be used, (2) the prevalence of Helicobacter pylori in the population should not be too low, and (3) an effective anti-H pylori regimen should be prescribed taking sufficient time to instruct and motivate the patient.
Management of motor neurone disease
Authors: Howard R S , Orrell R W
Published: January 2003

Motor neurone disease is a progressive neurodegenerative disorder leading to severe disability and death. It is clinically characterised by mixed upper and lower motor neurone involvement affecting bulbar, limb, and respiratory musculature. Recent guidelines have established diagnostic criteria and defined management of the condition. In a proportion of familial amyotrophic lateral sclerosis there is a mutation in the gene encoding the enzyme copper/zinc superoxide dismutase 1; this has allowed mutation screening and generated considerable laboratory based research. The diagnosis must be given with care and consideration and close follow up is essential. Management involves a multidisciplinary team based in the hospital and the community. Riluzole is the only drug shown to have a disease modifying effect and has been approved by the National Institute for Clinical Excellence. The essence of care is good symptomatic management, including nutritional support with percutaneous endoscopic gastrostomy and ventilatory care with non-invasive ventilation. Palliative care should be introduced before the terminal stages after careful discussion with the patient and carers. Knowledge of this condition has grown dramatically recently with a parallel improvement in treatment and ability to deal with the most troublesome problems.