bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibiotics that inhibit cell wall biosynthesis induce expression of the Bacillus subtilisσW and σM regulons
Authors: Cao Min , Wang Tao , Ye Rick , Helmann John D.
Published: March 2003

Bacillus subtilis
encodes seven extracytoplasmic function (ECF) sigma factors. The
σ
W
regulon includes functions involved in detoxification and protection against antimicrobials, whereas
σ
M
is essential for growth at high salt concentrations. We now report that antibiotics that inhibit cell wall biosynthesis induce both
σ
W
and
σ
M
regulons as monitored using DNA microarrays. Induction of selected
σ
W
‐dependent genes was confirmed using
lacZ
reporter fusions and Northern blot analysis. The ability of vancomycin to induce the
σ
W
regulon is dependent on both
σ
W
and the cognate anti‐
σ
, RsiW, but is independent of the transition state regulator AbrB. These results suggest that the membrane‐localized RsiW anti‐
σ
W
factor mediates the transcriptional response to cell wall stress. Our findings are consistent with the idea that one function of ECF
σ factors is to coordinate antibiosis stress responses and cell envelope homeostasis.
Large‐scale identification of serotype 4 Streptococcus pneumoniae virulence factors
Authors: Hava David L. , Camilli Andrew
Published: March 2003

Streptococcus pneumoniae
(the pneumococcus) is carried in the nasopharynx of healthy individuals, but can spread to other host sites and lead to pneumonia, bacteraemia, otitis media and meningitis. Although it is logical to think
a priori
that differential gene expression would contribute to the ability of this patho­‐gen to colonize different sites, in fact very few genes have been demonstrated to play tissue specific roles in virulence or carriage. Using signature‐tagged mutagenesis to screen 6149
mariner ‐transposon insertion strains, we identified 387 mutants attenuated for infection in a murine model of pneumonia. Among these mutants are ones with disruptions in a number of putative tissue‐specific transcriptional regulators, surface proteins, metabolic proteins and proteins of unknown function, most of which had not previously been associated with virulence. A subset of these, including most of those with insertions in putative transcriptional regulators, was examined for phenotypes in murine models of bacteraemia and nasopharyngeal carriage. Four classes of mutants defective in infection models of the: (I) lung, (II) lung and blood, (III) lung and nasopharynx, and (IV) all three tissues were identified, thus demonstrating the ex­‐istence of tissue‐specific pneumococcal virulence factors. Included in these strains were two with ­disruptions in a genetic locus that putatively codes for a transcriptional regulator, three surface proteins and three sortase homologues. Mutation analysis revealed that three of the seven genes in this locus are virulence factors that are specific to mucosal ­surfaces.
Two transcriptional regulators GlnR and GlnRII are involved in regulation of nitrogen metabolism in Streptomyces coelicolor A3(2)
Authors: Fink D. , Weißschuh N. , Reuther J. , Wohlleben W. , Engels A.
Published: March 2003

Streptomyces coelicolor
has an unusually large arsenal of glutamine synthetase (GS) enzymes: a prokaryotic GSI‐β‐subtype enzyme (encoded by
glnA
), three annotated
glnA
‐like genes of the GSI‐α‐subtype and a eukaryote‐like glutamine synthetase II (encoded by
glnII
). Under all tested conditions, GSI was found to represent the dominant glutamine synthetase activity. A significant heat‐labile GSII activity, which is very low to undetectable in liquid‐grown cultures, was only detected in morphologically differentiating
S. coelicolor
cultures. Analysis of
glnA
and
glnII
transcription by S1 nuclease mapping and
egfp
fusions revealed that, on nitrogen‐limiting solid medium,
glnII
but not
glnA
expression is upregulated. An OmpR‐like regulator protein, GlnR, has previously been implicated in transcriptional control of
glnA
expression. Gel retardation analysis revealed that GlnR is a DNA‐binding protein, which interacts with the
glnA
promoter. It is not autoregulatory and does not bind to the upstream regions of the
glnA
‐like genes of the α‐subfamily, nor to the
glnII
promoter
in vitro
. A second GlnR target was identified upstream of the
amtB
gene, encoding a putative ammonium transporter.
amtB
forms an operon with
glnK
(encoding a PII protein) and
glnD
(encoding a putative PII nucleotidylyltransferase) shown by S1 nuclease protection analysis and reverse transcription‐polymerase chain reaction (RT‐PCR). An
amtB
and
glnA
promoter alignment revealed a putative GlnR operator structure. Downstream of
glnII
, a gene encoding for another OmpR‐like regulator, GlnRII, was identified, with strong similarity to GlnR. Gel shifts with GlnRII showed that the promoters recognized by GlnR are also targets of GlnRII. However, GlnRII also interacted with the
glnII
upstream region. Only inactivation of
glnR
resulted in a glutamine auxotrophic phenotype, whereas the
glnRII mutant can grow on minimal medium without glutamine.
Modular domain structure in the Candida glabrata adhesin Epa1p, a β1,6 glucan‐cross‐linked cell wall protein
Authors: Frieman Matthew B. , McCaffery J. Michael , Cormack Brendan P.
Published: March 2003

The yeast pathogen Candida glabrata adheres avidly to cultured human epithelial cells. This interaction depends on the expression of EPA1, which encodes a lectin belonging to a large family of GPI‐anchored glucan‐cross‐linked cell wall proteins (GPI‐CWPs) found in diverse fungal species. To understand the relationship between different domains of EPA1 and its function, we have mapped functional domains of Epa1p and analysed their contribution to Epa1p function. We found that the N‐terminal third of the protein contains the ligand‐binding domain, and that the GPI anchor is essential both for cross‐linking in the cell wall and for Epa1p‐mediated adherence. We also found that the C‐terminal Ser/Thr‐rich domain, characteristic of many GPI‐CWPs, was absolutely essential for function. Although Epa1p derivatives lacking the Ser/Thr domain were expressed abundantly in the cell wall, they were localized to internal layers of the cell wall; such constructs were unable to mediate adherence. The outer layer of the yeast cell wall is known to act as a permeability barrier; we found that the C‐terminal Ser/Thr‐rich region was absolutely required to project the N‐terminal domain of Epa1p through this permeability barrier and into the external environment. Thus, the Ser/Thr‐rich domain of Epa1p and, presumably, of other related GPI‐CWPs serves an essential structural role in localization of the protein at the external surface of the yeast cell where it can interact with its ligand. In conclusion, Epa1p has a modular structure, with each domain serving a distinct and essential role in the function of the adhesin.
The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium‐mediated signalling
Authors: Serrano Raquel , Ruiz Amparo , Bernal Dolores , Chambers James R. , Ariño Joaquín
Published: March 2003

The short‐time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays. A total of 150 genes increased their mRNA level at least twofold within 45 min. Alkalinization resulted in the repression of 232 genes. The response of four upregulated genes, ENA1 (encoding a Na+‐ATPase also induced by saline stress) and PHO84, PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further. The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants. Mapping of the ENA1 promoter revealed two pH‐responsive regions. The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent. PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4. In contrast, part of the alkali‐induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506. Heterologous promoters carrying the minimal calcineurin‐dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH‐induced expression. These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response.
Genome plasticity in Acinetobacter: new degradative capabilities acquired by the spontaneous amplification of large chromosomal segments
Authors: Reams Andrew B. , Neidle Ellen L.
Published: March 2003

In Acinetobacter sp. ADP1, growth on benzoate requires regulation of the cat genes by two transcriptional activators. Here, mutants were obtained from a strain lacking both activators by selecting for growth on benzoate medium. The mutants, which arose within 3 weeks at a frequency of approximately 10−8, carried amplified chromosomal regions (amplicons) encompassing the cat genes. Multiple occurrences of low‐level expression of catA and the catBCIJFD operon provided sufficient transcription for growth. The amplicons of four independently isolated mutants varied in size from approximately 30–100 kbp of the normally 3.8 Mbp chromosome. Mutants had approximately 10–20 copies of an amplicon in adjacent head‐to‐tail orientations. At the amplicon's chromosomal endpoint, an atypical junction juxtaposed normally distant DNA regions from opposite sides of the cat genes. The sequences of these junctions revealed the precise recombination sites underlying amplification. Additionally, amplicon stability was evaluated in the absence of selective pressure. The natural competence of Acinetobacter for transformation by linear DNA has allowed the development of a powerful new model system for investigating chromosomal rearrangements and for engineering DNA amplifications for wide‐ranging applications. The frequent spontaneous amplification of these large chromosomal segments demonstrated the importance of supra‐operonic gene clustering in the evolution of catabolic pathways.
Swallows, oesophageal and gastric motility in normal subjects and in patients with gastro‐oesophageal reflux disease: a 24‐h pH‐manometric study
Authors: GROSSI  , CICCAGLIONE  , TRAVAGLINI  , MARZIO
Published: March 2003

Background: the motor aspects underlying gastro‐oesophageal reflux disease (GORD) are still not completely clear. Aim: to evaluate the relationship between oesophageal and gastric motility in GORD patients. Patients: twelve patients with grade I–II oesophagitis, mean age 45 yr, and 10 healthy subjects, mean age 42 yr, were studied. Methods: a pH‐manometry was performed to analyse oesophageal and gastric motility, swallows and oesophageal pH values for the whole 24‐h period, and for the 2‐min period before and after each reflux episode. Results: as compared to controls, GORD patients showed in the 24‐h period, a greater number of swallows (P < 0.01) and a lower percentage of post deglutitive propagated oesophageal body waves (P < 0.05). The number of migrating motor complexes (MMC) was similar in the two groups, with a lower amplitude of phase III gastric waves in GORD. During MMC reflux episodes were seen only in GORD patients. After refluxes an increase in swallows, simultaneous and secondary oesophageal waves were detected in GORD patients, with a reduction of primary peristalsis. Isolated gastric contractions preceded reflux episodes more frequently in GORD patients than in controls. Conclusions: GORD patients showed an increase in swallows with altered post‐deglutitive oesophageal motility and a reduced amplitude of gastric MMC. Moreover small contractions of gastric antrum are present before acid refluxes, suggesting a multifactorial pathogenesis of the disease.
Effect of TKS159, a novel 5‐hydroxytryptamine4 agonist, on gastric contractile activity in conscious dogs
Authors: HAGA  , SUZUKI  , SHIBA  , MOCHIKI  , MIZUMOTO  , ITOH
Published: March 2003

A novel 5‐hydroxytryptamine (5‐HT)4 receptor agonist, TKS159, {4‐amino‐5‐chloro‐2‐methoxy‐N‐[(2S,4S)‐1‐ethyl‐2‐hydroxymethyl‐4‐pyrrolidinyl] benzamide}, has recently been developed as a gastroprokinetic drug. Cisapride is already used clinically to increase gastric contractions. The stimulatory effects of TKS159 and cisapride on gastric contractions were examined using force transducers chronically implanted on the vagally denervated pouch (Heidenhain pouch) and the vagally innervated main stomach in conscious dogs. Contractile activity was analysed by computer and expressed as a motor index. Intravenous administration of TKS159 or cisapride significantly increased the motor index in both the main stomach and the Heidenhain pouch during the fed and fasted states. Pharmacological characterization in the fasted state revealed that the contraction‐stimulating activity of TKS159 and cisapride on the stomach was significantly inhibited by atropine, hexamethonium and a 5‐HT4 receptor antagonist, SDZ 205–557. Granisetron (a 5‐HT3 receptor antagonist) significantly inhibited cisapride‐induced, but not TKS159‐induced gastric contractions. The plasma motilin concentration was significantly increased after cisapride, but not after TKS159 injection. In conclusion, TKS159 has a contractile‐stimulating effect on both the innervated and the denervated stomach. It is likely that a cholinergic pathway and 5‐HT4 receptors are involved in producing the contractions, although other mechanisms cannot be excluded. Cisapride has almost the same characteristics, but the present findings suggest the involvement of motilin and 5‐HT3 receptors in the effects of cisapride.
Identification of the optimal parameters for recording cortical evoked potentials to human oesophageal electrical stimulation
Authors: HOBSON  , AZIZ  , FURLONG  , BARLOW  , BANCEWICZ  , Thompson
Published: March 2003

Cortical evoked potentials in response to stimulation of the oesophagus may prove to be a powerful technique for assessing the oesophageal afferent pathway in health and disease. However, in order to maximize the potential of this technique it is essential that the optimal parameters for recording oesophageal CEP are established. The aim was to determine the optimal parameters required to record reproducible CEP. CEP were recorded from the vertex in eight subjects (age range 23–44 years). Electrical stimulation was performed 5 cm above the lower oesophageal sphincter using a bipolar ring electrode at 0.2 Hz. Protocol 1: to determine the stimulation intensity which generates the largest amplitude and shortest latency, two runs of 50 stimuli were applied at increasing intensities. Protocol 2: to determine the number of stimuli for optimal signal to noise ratio, 10 runs of 50 stimuli were recorded. Individual runs were averaged. Protocol 3: to determine the optimal inter‐run interval, CEP evoked by 200 stimuli were averaged using randomly chosen inter‐run intervals. Protocol 4: CEP reproducibility using parameters determined from Protocols 1–3 was assessed in three subjects on three separate occasions. The results were as follows: Protocol 1; P1 latency was shortest and P1–N1 amplitude largest at an intensity of 75% above threshold. Protocol 2; optimal signal‐to‐noise was achieved by averaging four runs of 50 stimuli. Protocol 3; the optimal interstudy interval was 10 min. Protocol 4; highly reproducible CEP were obtained in all individuals. Using these optimal parameters, it is possible to obtain highly reproducible oesophageal CEP to ES which can now be used for clinical study.
Effects of sympathetic nerve stimulation on membrane potential in the circular muscle layer of mouse distal colon
Authors: SPENCER  , BYWATER  , KLEMM
Published: March 2003

Little is known about the effects of sympathetic nerve stimulation on the membrane potential of colonic smooth muscle. In the distal colon of the mouse, intracellular microelectrodes were used to record the effects of lumbar colonic (LCN) and intermesenteric nerve (IMN) stimulation on circular muscle membrane potential in vitro. A two‐compartment organ bath was used to selectively perfuse the colon and inferior mesenteric ganglion (IMG). In the presence of nifedipine (1–2 μM) (to the colonic compartment only), spontaneous depolarizations (myoelectric complexes, MCs) were recorded about every 4 min. MCs consisted of a prolonged burst of rapid oscillations in membrane potential (≈2 Hz) that were superimposed on a slow depolarization (mean amplitude 12 mV). Single electrical stimuli (600 μsec duration) delivered to the LCN or IMN did not elicit a detectable change in the membrane potential. However, trains of stimuli (e.g. 60 pulses at 10–20 Hz) to the LCN or IMN during the intervals between MCs evoked a depolarization (with superimposed action potentials in the absence of nifedipine). Trains of stimuli delivered during the plateau phase of the MC reduced or abolished the rapid oscillations, without a further membrane depolarization. The MC period was unaffected by stimulation of the IMN or LCN. Responses were abolished by the selective perfusion of guanethidine (1 μM) to the colon, or by severing the LCN. Hexamethonium (500 μM) (to the colon) abolished MCs, induced sustained depolarization and attenuated the amplitude of the sympathetic depolarizations by 74%. In hexamethonium, sympathetic responses remained attenuated when the membrane of the circular muscle was repolarised by sodium nitroprusside (1 μM). Immunohistochemical studies of the colon revealed intense immunoreactivity for tyrosine hydroxylase in the myenteric plexus but not in the circular muscle layer. It is suggested that responses to sympathetic nerve stimulation in the circular muscle layer of the mouse colon are secondary to actions on the enteric nervous system.
Short‐chain fatty acids present in the ileum inhibit fasting gastrointestinal motility in conscious pigs
Authors: CUCHE  , MALBERT
Published: March 2003

Colonic compounds, primarily short‐chain fatty acids (SCFAs), inhibit gastric tone in humans. However, since colonic compounds reflux into the ileum, SCFAs might act also at the ileal level. This study evaluates the effects of the contact of SCFAs with the ileum alone towards gastrointestinal motility in fasted conscious pigs. Gastrointestinal motility was recorded during ileal infusion of a mixture containing acetate, propionate and butyrate in intact vs ileostomized animals (n = 10). Ileal infusions of isomolar (500 mmol L−1) vs isocaloric (600 kJ L−1) SCFAs were also performed. SCFA mixture reduced significantly the amplitude and increased the frequency of antral contractions in intact (motility index: 2624 ± 503.4 vs 4077 ± 388.2) and ileostomized pigs (motility index: 2428 ± 678.1 vs 4709 ± 773.4) compared with iso‐osmotic saline. SCFAs at isomolar concentrations inhibited equally gastric motor pattern irrespective of their chemical structure. On the contrary, isocaloric concentrations of SCFAs induced graded effects: acetic acid being the most potent to reduce gastric motility. In conclusion, SCFAs inhibit gastrointestinal motility by a direct contact with the ileum. This inhibition was concentration dependent.
Increased tone of the rectal wall in response to feeding persists in patients with cauda equina syndrome
Authors: LEROI  , SAITER  , ROUSSIGNOL  , WEBER  , DENIS
Published: March 2003

The aim was to study fasting and postprandial rectal tone in patients with cauda equina injury. Electromechanical barostat measurement of rectal tone was made in 13 healthy volunteers and in five patients during a 10 min recording, while fasting and for 1 h after a 1000 kCal intake. A prompt decrease of rectal volume was observed in all control subjects and patients. The delay between the end of the meal and the onset of the rectal response was always less than 3 min in the five patients as well as in the control group. The rapidity of the rectal response to feeding observed in our five patients suggests that the rectal response was mediated via a neural or neurohumoral pathway despite severe injury of the sacral parasympathetic supply.
Motor activity of vascularly perfused rat duodenum. 2. Effects of VIP, PACAP27 and PACAP38
Authors: YAMAMOTO  , KUWAHARA  , FUJIMURA  , MAEDA  , FUJIMIYA
Published: March 2003

We examined the mechanisms of effects of vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP) 27 and PACAP38 on spontaneously occurring pressure waves in ex vivo perfused rat duodenum. VIP and PACAPs dose‐dependently reduced the percentage motor index of pressure waves; this reduction was not prevented by atropine, hexamethonium or tetrodotoxin (TTX). VIP and PACAPs abolished acetylcholine‐induced stimulation of pressure waves, even in the presence of TTX. These findings suggest that VIP and PACAPs may exert direct inhibitory effects via VIP/PACAP receptors located on smooth muscle rather than via cholinergic receptors. The inhibitory effects of VIP and PACAPs were partially antagonized by the VIP receptor antagonists VIP(10–28), suggesting that VIP and PACAPs share common receptor sites on intestinal smooth muscle. The effects of VIP and PACAPs were completely antagonized by nitric oxide (NO) synthase inhibitor NG‐nitro‐
L‐arginine (L‐NA), suggesting that NO mediates the inhibitory effects of VIP and PACAPs on duodenal motility. Furthermore, single injection of L‐NA stimulated spontaneously occurring pressure waves, while VIP(10–28) did not affect them. These findings suggest that VIP/PACAPs and NO strongly interact as an inhibitory mediator on duodenal motility, but that their modes of action in doing so may differ.
Temporal and spatial rhythmicity of jejunal wall motion in rats
Authors: Bouchoucha  , Benard  , Dupres
Published: March 2003

Isolated segments of jejunum of fasted rats exhibit regular rhythmic contractions at the same frequency as slow‐waves. The aim of the present study was to search for a possible spatial rhythmicity of this activity. Using a video imaging technique, jejunal segments of 50 rats were studied. Only experiments (n=76) with no propagated contractions at visual inspection were included in the study. After the measurement, a spectral analysis of the diameter variations was performed. The bands were characterized by four parameters: level, main frequency, amplitude and phase. At each level, the phase varied, suggesting that the same rhythmic phenomenon occurred, but with a delay as a function of the spatial position. In 58 measurements, the rhythmic activity had a frequency near 0.50 Hz and in 18, near 0.25 Hz. Phase difference was found in 32 segments (42%). The variation with distance was linear as a function of time and its length was greater for the low‐frequency group than for the high‐frequency group (25.6 ± 9.4 vs. 33.3 ± 5.2 mm, P=0.015). By contrast, the speed of propagation was not significantly different. The wavelength λ of the spatial rhythmicity was 27.7 ± 23.2 and 9.8 ± 4.2 mm (P=NS) in the high‐ and low‐frequency groups, respectively. This corresponds to a speed of propagation of v=λ*f, where f is the frequency of the wall motion (7.0 ± 5.2 vs. 5.2 ± 2.2 mm sec−1, P=NS).
Endomorphin‐1 and ‐2, endogenous ligands for the μ‐opioid receptor, inhibit striated and smooth muscle contraction in the rat oesophagus
Authors: Storr  , Geisler  , Neuhuber  , Schusdziarra  , Allescher
Published: March 2003

Recently, morphological evidence for an interaction of autonomic nerve fibres and extrinsic motor innervation of the rat oesophagus has emerged. The aim of the present study was to investigate the possible influence of endogenous and exogenous opioids on rat oesophageal smooth and striated muscle function in vitro. The entire oesophagus (excluding the lower oesophageal sphincter) with both Nervi (Nn) vagi, including the Nn recurrentes, was dissected and placed in an organ bath (100 mL, 37°) with oxygenated Krebs–Ringer buffer. Contractile activity was measured in a longitudinal direction with a force transducer. Both Nn vagi were placed on a bipolar platinum electrode 2 cm distant from the oesophagus. Vagal stimulation (VS), applied for 1 s (40 V, 0.5 ms, 20 Hz) resulted in a biphasic contractile response that was completely blocked by 10–6 M tetrodotoxin. The first part consisted of a tetanic striated muscle contraction, as it was abolished by tubocurarine (10–5 M, n=5) but unaffected by atropine (10–6M, n=3) or hexamethonium (10–4 M, n=4). In contrast, the second part was completely inhibited by hexamethonium (10–4 M) and atropine (10–6 M), whereas tubocurarine (10–5 M) showed no influence, indicating a stimulation of preganglionic nerve fibres supplying oesophageal smooth muscle (muscularis mucosae) via relays in myenteric ganglia. In order to characterize opioid influence on the oesophageal striated and smooth muscle contractility, the following experiments were carried out. 10–6 M endomorphin‐1 and ‐2, endogenous μ‐opioid‐receptor agonists, reduced the contractile response of the striated (EM‐2, −25.1 ± 5.3%; n=16), and the smooth muscle (EM‐2, – 81.9 ± 3.3%; n=11). Both effects were reversible by the opioid receptor antagonist naloxone (10–6 M) and therefore, mediated via opioid receptors. Neither SNC‐80, an agonist on the δ‐opioid‐receptor, U‐69593, an agonist on the κ‐opioid‐receptor, nor nociceptin, an agonist at the ORL1 (opioid receptor‐like1) receptor, had a significant effect on the striated muscle contraction. In contrast to SNC‐80, U‐69593 and nociceptin inhibited smooth muscle contraction but this relaxation could not be antagonized by naloxone. None of the opioid receptor antagonists used had an effect on basal tonus or muscle contraction following VS. Our data provide evidence for an autonomic modulation of vagal motor innervation of the striated and smooth oesophageal muscle. Endomorphin‐1 and ‐2, both selective μ‐opioid receptor agonists, cause an inhibition of striated and smooth muscle response which is reversible by naloxone, an opioid receptor antagonist. The location of the μ‐opioid receptor still has to be established.
Abnormal cerebral processing of oesophageal stimuli in patients with noncardiac chest pain (NCCP)
Authors: Hollerbach S. , Bulat R. , May A. , Kamath M. V. , Upton A. R. M. , Fallen E. L. , Tougas G.
Published: March 2003

In noncardiac chest pain (NCCP), altered visceral perception may result from abnormal cerebral processing of sensory input rather than abnormalities of afferent pathways. However, the interactions between symptoms, autonomic function and oesophageal stimuli are poorly studied. Oesophageal stimulation elicits reproducible cortical evoked potentials [CEP] and modulates heart rate variability via vagal pathways, as visible on power spectrum analysis of heart rate variability [PS‐HRV]. These methods are increasingly used to study the function of visceral afferent neural pathways in human. The aim of this study was to compare EP and PS‐HRV during oesophageal stimuli in NCCP and controls. Twelve healthy volunteers (one female, 11 male; aged 24–51 years; mean 32 ± 8 years), and eight NCCP patients (three female, five male; age range 26–58, mean 40.5 ± 10 years) were studied. Electrical oesophageal stimulation (EOS; 200 μs, 0.2 Hz, 25 stimuli) was applied to the oesophageal wall 5 cm above the lower oesophageal sphincter (LOS), and perception thresholds (measured in mA) determined. EP responses were recorded using 22 standard electroencephalogram scalp electrodes. Autonomic activity was assessed using PS‐HRV, before, during, and after oesophageal stimulation. Measured PS‐HRV indices included high frequency (HF; 0.15–0.5 Hz) and low frequency (LF; 0.06–0.15 Hz) power, respectively, assessing vagal and sympathetic activity, and the LF/HF ratio. EOS perception occurred at lower thresholds in NCCP than in controls (3.6 ± 1 vs. 7.8 ± 2 mA, P < 0.05). EP amplitude was greater (13 ± 2 vs. 6 ± 1 μV, P < 0.0001), and latency longer in controls vs. NCCP (191 ± 7 ms vs. 219 ± 6 ms, P < 0.001). In NCCP, EOS decreased sympathetic outflow (low frequency peak on PS‐HRV) and increased cardiovagal activity (high frequency peak, P < 0.02) to a significantly higher degree in comparison with controls. During EOS, heart rate decreased in NCCP from 68 vs. 62 beats min–1 (P < 0.003) but not in controls. In NCCP patients, EOS was perceived at lower intensities and was associated with a greater cardiovagal reflex response. EP responses associated with EOS were smaller in NCCP than in controls, suggesting that an increased perception of oesophageal stimuli results from an enhanced cerebral processing of visceral sensory input in NCCP, rather than from hyperalgesic responses in visceral afferent pathways.
Secretin induces variable inhibition of motility in different parts of the Australian possum sphincter of Oddi
Authors: Al‐Jiffry B. O. , Jobling J. M. , Schloithe A. C. , Toouli J. , Saccone G. T. P.
Published: March 2003

The sphincter of Oddi (SO) may not function as a single structure. We aimed to determine the response of the proximal and distal segments of the bile duct (BD‐SO) and pancreatic duct (PD‐SO) components of the SO to secretin, with and without neural blockade with tetrodotoxin (TTX). In anaesthetized Australian possums, separate manometry catheters were placed in the proximal and distal BD‐SO or PD‐SO segments to record motility. Secretin, 50–1000 ng kg–1, was administered, followed by TTX, and re‐administration of secretin, 500 and 1000 ng kg–1. Changes in the motility index (MI, frequency × mean amplitude) were determined. Statistical analysis utilized repeated‐measures ANOVA. Secretin produced a dose‐dependent decrease in MI from the proximal and distal BD‐SO and PD‐SO (all P < 0.001). The maximum inhibition, at 1000 ng kg–1, was 21 ± 4%, 33 ± 6% and 42 ± 5% of control (mean ± SEM), for proximal and distal BD‐SO, and distal PD‐SO, respectively. The proximal PD‐SO MI, however, was inhibited to 62 ± 6% of control, at 1000 ng kg–1. TTX enhanced the secretin‐induced response to the same level at the four sites (P < 0.02). We conclude that secretin inhibits the motility of the possum SO in a nonuniform manner and is modulated by neural activity.
Relaxation of canine gallbladder to nerve stimulation involves adrenergic and non‐adrenergic non‐cholinergic mechanisms
Authors: Alcón S. , Morales S. , Camello P. J. , Salido G. M. , Miller S. M. , Pozo M. J.
Published: March 2003

Electrical field stimulation (EFS) of dog gallbladder strips induced a frequency‐dependent contractile response followed by an off‐relaxation that was turned into a pure inhibitory response after atropine pretreatment. Guanethidine reduced the atropine‐induced relaxing responses, so an adrenergic mechanism can partially account for the nerve‐mediated gallbladder relaxation. However, guanethidine pretreatment also revealed a nonadrenergic noncholinergic (NANC) relaxation induced by EFS, which was frequency independent. NANC relaxations were reduced by L‐arginine methyl ester (L‐NAME, 100 μmol L–1), a nitric oxide synthase inhibitor (D‐p‐Cl‐Phe6, Leul7; 10 μmol L–1), a vasoactive intestinal peptide (VIP) receptor antagonist, and an inhibitor of haem oxygenase, (copper protoporphyrin IX; CuPP‐IX; 10 μmol L–1), suggesting that nitric oxide (NO), VIP and carbon monoxide (CO), respectively, are released in response to EFS. Immunoreactivities for haem oxygenase‐2 (HO‐2) and VIP, and histochemical staining for NADPH diaphorase were observed in nerve cell bodies and fibres, demonstrating the presence of CO, VIP and NO as putative NANC neurotransmitters in dog gallbladder. These data support the hypothesis that NO, VIP and CO contribute to NANC relaxation of the canine gallbladder.
Propagating contractions of the circular muscle evoked by slow stretch in flat sheets of guinea‐pig ileum
Authors: Brookes S. J. H. , D'Antona G. , Zagorodnyuk V. P. , Humphreys C. M. S. , Costa M.
Published: March 2003

Flat sheet preparations of guinea‐pig ileum were stretched circumferentially and the propagation of circular muscle contractions along the preparation was investigated. Slow stretch, at 100 μm s–1, of a 50‐mm long flat sheet of intestine, evoked circular muscle contraction orally, which propagated, without decrement, for up to 30 mm. This occurred despite circular muscle shortening being prevented, and in the absence of propulsion of contents. Thus, propagation in this flat sheet preparation could not explained on the basis of neuro‐mechanical interactions, as previously proposed. Irrespective of the length of preparations, contraction amplitude decreased significantly in the most aboral 10–15 mm of intestine. This was not due to descending inhibitory pathways, but was associated with interruption of ascending excitatory pathways near the aboral end. Slow waves were not detected in circular muscle cells in any preparation (n=8). Smooth muscle action potentials evoked in circular muscle cells, in the presence of tetrodotoxin (TTX, 0.6 μmol L–1), did not propagate for more than 1 mm in the longitudinal axis. Propagation of circular muscle activity, evoked by slow stretch of flat sheet preparations, reveals the presence of a mechanism other than myogenic spread or the neuro‐mechanical interactions previously proposed to account for propagation; the nature of this mechanism remains to be determined.
Intestinal motor stimulation by the 5‐HT4 receptor agonist ML10302: differential involvement of tachykininergic pathways in the canine small bowel and colon
Authors: De Ponti F. , Crema F. , Moro E. , Nardelli G. , Croci T. , Frigo G. M.
Published: March 2003

5‐Hydroxytryptamine (5‐HT)4 receptor agonists stimulate gut motility through cholinergic pathways, although there are data suggesting that noncholinergic (tachykininergic) excitatory pathways may also be involved. Differences may exist between the small bowel and colon. Our aims were: (i) to compare the prokinetic effect exerted by the 5‐HT4 receptor agonist ML10302 in the canine small bowel and colon in vivo; and (ii) to investigate the role of tachykininergic pathways in mediating this response. In fasting, conscious dogs, chronically fitted with electrodes and strain‐gauge force transducers along the small bowel and colon, intravenous injection of ML10302 (35 μg kg–1) immediately stimulated spike activity and significantly increased propagated myoelectrical events at both intestinal levels. In the small bowel, the effects of ML10302 were unchanged by previous administration of the selective NK1 tachykinin receptor antagonist SR140333, the NK2 tachykinin receptor antagonist SR48968, or the NK3 tachykinin receptor antagonist SR142801. In the colon, all tachykinin receptor antagonists significantly inhibited stimulation of spike and mechanical activity by ML10302, without affecting ML10302‐induced propagated myoelectrical events. Atropine (100 μg kg–1 i.v.) suppressed the stimulatory effect of ML10302 at both intestinal levels. In conclusion, the 5‐HT4 receptor agonist ML10302 induced significant prokinesia both in the small bowel and colon through activation of cholinergic pathways. Tachykininergic pathways are not involved in the ML10302‐induced prokinesia in the small bowel, but they play an important role in mediating the colonic motor response to ML10302.
Automated, quantitative analysis of interdigestive small intestinal myoelectric activity in rats
Authors: VAN SCHELVEN L. J. , NIEUWENHUIJS V. B. , AKKERMANS L. M. A.
Published: March 2003

Quantitative analysis of myoelectric activity (EMG), to investigate small intestinal motility in rats, is normally based on manual classification into sequences of phase I, phase II and phase III. This classification is partly subjective. We aimed to develop a more objective method for the analysis of the migrating myoelectric complex (MMC). From the EMG, a derived signal is calculated as a measure of activity. Depending on the level of this derived signal, the EMG is classified into `quiescent phase', `irregular phase' or `activity front'. The threshold levels for these phases are automatically calculated from the EMG data. A proposal for subdivision into MMCs is automatically generated. To calculate MMC length, the user must manually reject nonpropagated activity fronts. While developing the method, more than 19 derived signals were tested. These included variants of spike frequency, signal power and spike‐burst length. The spike frequency signal was chosen because it gave minimal deviation from manual classification. Using the new automated method, recordings from the jejunum of 15 healthy rats were analysed (6 h each). The calculated phase lengths were consistent with the results of manual analysis. The presented method allows objective analysis of the interdigestive EMG signals of the small intestine.
The 13C‐octanoic acid breath test: validation of a new noninvasive method of measuring gastric emptying in rats
Authors: Schoonjans R. , Van Vlem B. , Van Heddeghem N. , Vandamme W. , Vanholder R. , Lameire N. , Lefebvre R. , De Vos M.
Published: March 2003

Abstract
 
Currently available rat models for measuring gastric emptying are hampered by the necessity to kill the animals at the end of each experiment, which makes repetitive testing impossible. We have developed and validated a noninvasive test model, adapted from the13C‐octanoic breath test in humans, for repetitive measurements of gastric emptying in rats. Male Wistar rats were trained on a fixed protocol to eat a piece of pancake doped with 1 μg13C‐octanoic acid after 12 h fasting, and to stay thereafter in cylindrical glass cages. Breath tests were performed by a fully automated system of computer‐guided switching valves, which collected consecutive breath samples. All breath samples were analysed by gas chromatography and isotope mass spectrometry. The area under the curve (AUC) from the cumulative13CO2excretion from 0 to 6 h was determined by the trapezium method to calculate the gastric half‐emptying times (t½). Inter‐day variability was determined. The effect of subcutaneous or intraperitoneal injection of saline was studied. The test was further validated for pharmacological interventions by oral administration of cisapride and parenteral administration of atropine, to induce, respectively. acceleration and delay of gastric emptying. Mean gastric emptying times ± SD of 24 rats were 119.3 ± 28.2 min, 138.7 ± 26.0 min, and 124.5 ± 30.9 min on three different test days. The mean coefficient of variation of three repeated measurements in the same 24 rats was 17.5%. No significant differences were observed after subcutaneous or intraperitoneal injection of saline. In a second test series of eight rats, cisapride significantly accelerated gastric emptying (mean t½ 112.7 ± 33.1 min, P < 0.05), while atropine caused a significant delay (mean t½ 205.9 ± 24.9 min, P < 0.05) when compared to control test results (mean t½ 140.7 ± 16.7 min) in the same rats. We validated the13C‐octanoic breath test to study gastric emptying in rats. This test method obviates the necessity to kill laboratory animals and allows repetitive measurements of gastric emptying to study its physiology or pathophysiology as well as the effect of pharmacological agents.
Influence of naloxone on gastric emptying of solid meals, myoelectrical gastric activity and blood hormone levels in young healthy volunteers
Authors: Reber P. U. , Netzer P. , Gaia C. , Wildi S. , Lourens ST. , Noelpp U. , Lüscher D. , Varga L. , Brenneisen R. , Scheurer U.
Published: March 2003

Abstract
 There is considerable evidence that opioid mechanisms are involved in the mediation of pyloric motor responses that in turn regulate gastric emptying. The purpose of this randomized, placebo‐controlled crossover study was to investigate the effect of naloxone on gastric emptying of a solid meal, gastric myoelectrical activity and the postprandial release of gastrointestinal peptides and neuropeptides in 20 healthy volunteers. Naloxone was administered as an intravenous bolus, followed by continuous infusion according to an intravenous dosing nomogram. Gastric emptying time was evaluated by scintigraphy and gastric myoelectrical activity was evaluated by cutaneous electrogastrography. Naloxone did not significantly alter gastric half‐emptying time and postprandial dominant gastric electrical frequency compared with placebo. It also did not significantly change the plasma levels of several peptide hormones with the exception of neuropeptide Y, which was significantly increased (P = 0.001). In conclusion, in doses that influence human intestinal motility, naloxone had no effect on gastric motility and release of several peptide hormones in healthy male volunteers. The importance of the isolated increased neuropeptide Y plasma level needs further investigation.
Widespread occurrence of argyrophilic glial inclusions in Parkinson's disease
Authors: Hishikawa N. , Hashizume Y. , Yoshida M. , Sobue G.
Published: March 2003

Argyrophilic glial inclusions, which are immunohistochemically positive for α‐synuclein but negative for tau protein, were examined in the brain of Parkinson's disease (PD) patients. Autopsied brains of 10 individuals who died from PD, of two incidental Lewy body disease cases and of five age‐matched individuals whose deaths were caused by non‐neurological diseases were studied, histopathologically, by Gallyas‐Braak staining and, immunohistochemically, with anti‐α‐synuclein antibody, anti‐ubiquitin, and anti‐tyrosine hydroxylase. All postmortem PD brains showed a significant number of argyrophilic glial inclusions, but no glial inclusions were found in control brains. The inclusions were found not only in the regions showing neuronal loss and gliosis, such as the substantia nigra, locus ceruleus and dorsal vagal nucleus, but also in regions without neuronal loss and gliosis, such as the cerebral cortex, cerebral white matter, striatum, globus pallidus, thalamus, cerebellum and spinal cord. The distribution and density of glial inclusions in PD brains varied from case to case but, in the cerebral cortex, the number of glial inclusions were fairly well correlated with the number of Lewy bodies. The distribution pattern of glial inclusions also showed a striking resemblance to that of catecholaminergic neurones and fibres. The abnormal accumulation of α‐synuclein in glial cells was more widespread than neurone loss, and appears to be an important pathological feature of PD.
Immunoreactivity for Bcl‐2 and C‐Jun/AP1 in hippocampal corpora amylacea after ischaemia in humans
Authors: Botez G. , Rami A.
Published: March 2003

Corpora amylacea (CAm) are regarded as a hallmark of brain ageing, but little is known about their role in normal and pathological circumstances. CAm contain, in addition to glucose polymers, ageing‐, stress‐ and proinflammatory proteins. In view of their almost universal occurrence and their cumulation with time, formation of CAm may represent a basic mechanism for the management of metabolic degradation products. In this context, we studied samples from post‐mortem cases with repetitive brain hypoxic episodes in the past history. We investigated, by immunohistochemistry, the presence of Bcl‐2, c‐jun and bax in CAm. CAm showed immunoreactivity for the mitochondrial membrane associated protein Bcl‐2, and for the major component of activator protein 1 transcriptional factor c‐Jun. We found higher numbers of CAm in the hippocampus and the dentate gyrus in cases with repetitive cerebral hypoxia than in controls. We conclude that: (1) the presence of C‐Jun and Bcl‐2 within the glucose polymer mass of CAm may be related to mitochondrial damage and/or a transient overload of proteolytic systems during cellular injury; and (2) repetitive cellular stress during life may cause the age‐related increase of CAm in elderly subjects.
Quantification of vacuolation (‘spongiform change’), surviving neurones and prion protein deposition in eleven cases of variant Creutzfeldt–Jakob disease
Authors: Armstrong R. A. , Cairns N. J. , Ironside J. W. , Lantos P. L.
Published: March 2003

Vacuolation (‘spongiform change’) and prion protein (PrP) deposition were quantified in the cerebral cortex, hippocampus, dentate gyrus and molecular layer of the cerebellum in 11 cases of variant Creutzfeldt–Jakob disease (vCJD). The density of vacuoles was greater in the cerebral cortex compared to the hippocampus, dentate gyrus and cerebellum. Within the cortex, vacuole density was significantly greater in the occipital compared to the temporal lobe and the density of surviving neurones was greatest in the occipital lobe. The density of the non‐florid PrP plaques was greater in the cerebellum compared to the other brain areas. There were significantly more florid‐type PrP plaques in the cerebral cortex compared to the hippocampus and the molecular layer of the cerebellum. No significant correlations were observed between the densities of the vacuoles and the PrP plaques. The densities of vacuoles in the parietal cortex and the non‐florid plaques in the frontal cortex were positively correlated with the density of surviving neurones. The densities of the florid and the non‐florid plaques were positively correlated in the parietal cortex, occipital cortex, inferior temporal gyrus and dentate gyrus. The data suggest: (i) vacuolation throughout the cerebral cortex, especially in the occipital lobe, but less evident in the hippocampus and molecular layer of the cerebellum; (ii) the non‐florid plaques are more common than the florid plaques and predominate in the molecular layer of the cerebellum; and (iii) either the florid plaques develop from the non‐florid plaques or both types are morphological variants resulting from the same degenerative process.
Do quantitative or qualitative histological changes occur in the premotor substrate for vertical gaze as a function of ageing?
Authors: Henson C. , Staunton H. , Brett F. M.
Published: March 2003

Introduction:  Currently, there is debate in the clinical literature as to whether defects in vertical gaze occur as a consequence of normal ageing or a component of an underlying neurodegenerative disorder (Waite et al. Arch Neurol 1996; 53: 498). Although pathological changes have been demonstrated in diseased subjects (Brett et al. Arch Neurol 2001 in press), no study to date has addressed the question of normal ageing effects. The goal of our study was qualitatively and quantitatively to investigate histological changes in the primary premotor substrate of vertical gaze in young and aged subjects.

Subjects and methods:  Twenty‐three subjects were chosen from a group of 52 neurologically and pathologically normal subjects, ages 18–91. Using an unbiased, frame‐based sampling method (Gundersen et al. J Microscopy 1977; 111: 219–222), we quantified neuronal and glial cell densities in 10 young (<50 years old) and 13 aged (>65 years old) subjects in the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF), i.e. the key premotor substrate in the vertical gaze pathway.

Results:  We found no statistically significant difference in neuronal density (P ≥ 0.05), glial cell density (P ≥ 0.05) or neuron‐to‐glial cell ratios (P ≥ 0.05) between the young and the aged subjects.

Conclusion:  Our results support the conclusion that neuronal loss, neuronal atrophy or gliosis in the riMLF are not consequences of normal ageing.