bluuebunny/crossref_metadata_2025 huggingface dataset:
The switch from inorganic to organic sulphur assimilation in Escherichia coli: adenosine 5′‐phosphosulphate (APS) as a signalling molecule for sulphate excess
Authors: Bykowski Tomasz , Ploeg Jan R. van der , Iwanicka‐Nowicka Roksana , Hryniewicz Monika M.
Published: March 2003

The utilization of organosulphur compounds as sources of sulphur by Escherichia
coli is strongly repressed by sulphate. To search for the signal enabling E.
coli to alternate gene expression according to the sulphur source, we investigated
the transcriptional control of the ssuEADCB operon, required for the transport
and desulphonation of aliphatic sulphonates. We demonstrate that, of the two LysR‐type
regulators involved in expression from the ssu promoter, Cbl acts as a direct
and sufficient activator of transcription in vivo and in vitro, whereas
CysB downregulates the promoter efficiency. Most importantly, the Cbl‐mediated transcription
initiation at the ssu promoter in vitro is abolished in the presence
of an early metabolite of the sulphate assimilatory pathway, adenosine 5′‐phosphosulphate
(APS). This role for APS was confirmed in vivo by measuring the expression
of β‐galactosidase from a transcriptional ssu–lacZ fusion in strains containing different mutations blocking the synthesis and consumption of APS. Our data comprise the first evidence that APS may act as the negative cofactor of the transcriptional regulator Cbl, and that APS, and not sulphate itself, serves as the signalling molecule for sulphate excess.
Mapping the DNA‐binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI
Authors: Sheldon Paul J. , Busarow Sara B. , Hutchinson C. Richard
Published: March 2003

Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti‐biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA‐binding domains of the SARPs and the C‐terminal DNA‐binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR‐binding domain as a template, DnrI was mapped by truncation and site‐directed mutagenesis. Several highly conserved residues within the N‐terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the −35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG–dpsABCD promoter were performed by site‐directed mutage‐nesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG–dpsABCD operon.
ClpP‐dependent degradation of PopR allows tightly regulated expression of the clpP3 clpP4 operon in Streptomyces lividans
Authors: Viala Julie , Mazodier Philippe
Published: March 2003

Five clpP genes have been identified in Streptomyces coelicolor.

The clpP1 and clpP2 genes form one operon, the clpP3 and clpP4

genes form another, and clpP5 is monocistronic. Previous studies in Streptomyces

lividans have shown that the first operon (clpP1 clpP2) is required for

a normal cell cycle. Expression of the second operon (clpP3 clpP4) is activated

by PopR if the first operon is nonfunctional. We show here that PopR degradation

is primarily dependent on ClpP1 and ClpP2, but can also be achieved by ClpP3 and

ClpP4. The carboxy‐terminus of PopR plays an essential part in the degradation process.

Indeed, replacement of the last two alanine residues by aspartate residues greatly

increased PopR stability. These substitutions did not impair PopR activity and, as

expected, accumulation of the mutant form of PopR led to very strong expression of

the clpP3 clpP4 operon. Increased PopR levels led to delayed sporulation. The results obtained in this study support the notion of cross‐processing between ClpP1 and ClpP2.
Peptidyl‐tRNA hydrolase in Bacillus subtilis, encoded by spoVC, is essential to vegetative growth, whereas the homologous enzyme in Saccharomyces cerevisiae is dispensable
Authors: Menez Jeanne , Buckingham Richard H. , Zamaroczy Miklos de , Campelli Celine Karmazyn
Published: March 2003

Peptidyl‐tRNA hydrolase in Escherichia coli, encoded by pth, is essential for recycling tRNA molecules sequestered as peptidyl‐tRNA as a result of pre‐mature dissociation from the ribosome during translation. Genes homologous to pth are present in other bacteria, yeast and man, but not in archaea. The homologous gene in Bacillus subtilis, spoVC, was first identified as a gene involved in sporulation. A second copy of spoVC, under the control of the xyl promoter, was integrated into B. subtilis at the amy locus. In this background, interruption of the original gene was possible provided that expression of the copy at the amy locus was induced. When spoVC was interrupted, both vegetative growth and sporulation were dependent on xylose, showing that SpoVC is essential. The role of SpoVC in sporulation is discussed and appears to be consistent with previous hypotheses that a relaxation of translational accuracy may occur during sporulation. The homologous gene in Saccharomyces cerevisiae, yHR189W, has been interrupted in both haploid and diploid strains. The mutant haploid strains remain viable, as do the yHR189W mutant spores obtained by tetrad dis‐section, with either glucose or glycerol as carbon source, showing that the yHR189W gene product is dispensable for cell growth and for mitochondrial respiration.
Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria, a bladder colonization factor of uropathogenic Proteus mirabilis
Authors: Li Xin , Lockatell C. Virginia , Johnson David E. , Mobley Harry L. T.
Published: March 2003

Proteus mirabilis
is a common cause of urinary tract infection (UTI) in individuals with structural abnormalities or long‐term catheterization. The expression of mannose‐resistant/
Proteus
‐like (MR/P) fimbria is phase variable because of the inversion of a 251 bp DNA fragment that carries the promoter for the
mrp
operon. Previous studies have shown that
mrpI
, which is transcribed divergently from the
mrp
operon, encodes a recombinase capable of switching the orientation of this invertible element. In this study, we constructed isogenic
mrpI
null mutants from a clinical isolate of
P. mirabilis
, HI4320. A polymerase chain reaction (PCR)‐based invertible element assay revealed that the isogenic
mrpI
null mutants were locked in one phase, either expressing (locked on) MR/P fimbriae or not (locked off), which indicated that MrpI was the sole recombinase that regulated the phase variation of MR/P fimbria. The locked‐on and locked‐off mutants were evaluated for virulence in the CBA mouse model of ascending UTI by co‐challenges with each other and with the wild‐type strain. Results from these experiments demonstrated conclusively that the MR/P fimbria was a critical bladder colonization factor of uropathogenic
P. mirabilis and also suggested that the ability to switch off the expression of MR/P fimbria might be important for kidney colonization.
Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon
Authors: Baichoo Noel , Wang Tao , Ye Rick , Helmann John D.
Published: March 2003

The Bacillus subtilis ferric uptake repressor (Fur) protein coordinates a global transcriptional response to iron starvation. We have used DNA microarrays to define the Fur regulon and the iron starvation stimulon. We identify 20 operons (containing 39 genes) that are derepressed both by mutation of fur and by treatment of cells with the iron chelator 2,2′‐dipyridyl. These operons are direct targets of Fur regulation as judged by DNase I footprinting. Analyses of lacZ reporter fusions to six Fur‐regulated promoter regions reveal that repression is highly selective for iron. In addition to the Fur regulon, iron starvation induces members of the PerR regulon and leads to reduced expression of cytochromes. However, we did not find any evidence for genes that are directly activated by Fur or repressed by Fur under iron‐limiting conditions. Although genome searches using the 19 bp Fur box consensus are useful in identifying candidate Fur‐regulated genes, some genes associated with Fur boxes are not demonstrably regulated by Fur, whereas other genes are regulated from sites with little apparent similarity to the conventional Fur consensus.
Transcriptome analysis of all two‐component regulatory system mutants of Escherichia coli K‐12
Authors: Oshima Taku , Aiba Hirofumi , Masuda Yasushi , Kanaya Shigehiko , Sugiura Masahito , Wanner Barry L. , Mori Hirotada , Mizuno Takeshi
Published: March 2003

We have systematically examined the mRNA profiles of 36 two‐component deletion mutants, which include all two‐component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria–Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two‐component systems. A high correlation coefficient of expression profile was found between several two‐component mutants. Together, these results support the view that a network of functional interactions, such as cross‐regulation, exists between different two‐component systems. The compiled data are avail‐able at our website (http:ecoli.aist‐nara.ac.jpxpanalysis 2components).
Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins
Authors: Logan Susan M. , Kelly John F. , Thibault Pierre , Ewing Cheryl P. , Guerry Patricia
Published: March 2003

Flagellin from Campylobacter coli VC167 is post‐translationally modified at ≥ 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7‐diacetamido‐3,5,7,9 ‐ tetradeoxy ‐ l ‐ glycero ‐ l ‐ manno ‐ nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81‐176. VC167 lacked two modi‐fications present in 81‐176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81‐176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81‐176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS‐PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81‐176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.
Characterization of Exc, a novel protein required for the excision of Bacteroides conjugative transposon
Authors: Sutanto Yuri , Shoemaker Nadja B. , Gardner Jeffrey F. , Salyers Abigail A.
Published: March 2003

Conjugative transposons are integrated elements that excise from the chromosome, then transfer by conjugation to a recipient in which they integrate once again. Recently, a gene, designated exc, was shown to be essential for excision of the Bacteroides conjugative transposon (CTnDOT) from the chromosome. The deduced amino acid sequence of Exc had low amino acid sequence similarity to DNA topoisomerase III, an enzyme that relaxes DNA supercoils. This similarity raised the question of whether Exc protein was a topoisomerase and, if so, whether topoisomerase activity might contribute to the excision process. Here, we demonstrate that Exc does have topoisomerase activity in vitro. Exc relaxed supercoiled DNA, had a conserved tyrosine as its active site and required magnesium ions for its relaxation activity. However, although mutation of the catalytic tyrosine of Exc to phenylalanine abolished the ability of the enzyme to relax DNA supercoils in vitro, the mutation did not abolish the ability of the protein to mediate excision in vivo. This surprising result suggests that CTnDOT excision does not rely on the topoisomerase activity of Exc in vivo.
Strand‐specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriC
Authors: Weigel Christoph , Seitz Harald
Published: March 2003

We analysed the enzymatic activity (strand dis‐placement) of the Escherichia coli DnaB helicase on a mirror‐image pair of oligonucleotide‐based substrates mimicking the unwound replication origin oriC. Loading of the helicase complex occurred exclusively to the single‐stranded ‘lower strand’ part of the substrates. Full helicase activity required DnaA bound to the double‐stranded part of the substrates (oriC DnaA box R1) and to their single‐stranded ‘upper strand’ part. We assume that in vivo DnaA also loads the first of two helicase complexes – required for the assembly of two replication forks – to the lower strand of oriC during initiation of bidirectional chromosome replication in E. coli.
SycE allows secretion of YopE–DHFR hybrids by the Yersinia enterocolitica type III Ysc system
Authors: Feldman Mario F. , Müller Simone , Wüest Esther , Cornelis Guy R.
Published: March 2003

The Ysc type III secretion system allows Yersinia enterocolitica to translocate virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Some of the Yop effectors possess an individual chaperone called a Syc protein. The first 15 amino acids of the YopE effector constitute a secretion signal that is sufficient to promote secretion of several reporter proteins. Residues 15–50 of YopE comprise the minimal binding domain for the SycE chaperone. In this study, we investigated the secretion by the Ysc system of several YopE–DHFR hybrid proteins with different folding properties, and evaluated the role of SycE, the cognate chaperone of YopE, in this context. We have analysed the secretion of hybrids containing 16 (YopE16), 52 (YopE52) and 80 (the complete region covered by the chaperone, YopE80) amino acids of YopE or full‐length YopE (YopEFL) with wild‐type DHFR and two mutants with altered folding properties. The hybrids containing DHFRΔ77, the mutant whose folding properties are the most drastically affected, could be secreted in all the conditions tested, even in the absence of the chaperone SycE. In contrast, DHFRwt could only be secreted fused to the first 52 amino acids of YopE, and its secretion was strictly dependent on SycE. The hybrids YopE80–DHFRwt and YopEFL–DHFRwt were not secreted. YopEFL–DHFRwt completely jammed the channel in an SycE‐dependent fashion. Our experiments indicate that, in order to be secreted, proteins must be unfolded or only partially folded, and that TSS chaperones could keep their substrates in a secretion‐competent conformation, probably by preventing their folding. In addition, they show that the secretion apparatus can reject folded proteins if they are not deeply engaged into the injectisome.
Distribution and function of new bacterial intein‐like protein domains
Authors: Amitai Gil , Belenkiy Olga , Dassa Bareket , Shainskaya Alla , Pietrokovski Shmuel
Published: March 2003

Hint protein domains appear in inteins and in the C‐terminal region of Hedgehog and Hedgehog‐like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein‐splicing of their host‐protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N‐terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein‐like (BIL) domains. Bacterial intein‐like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C‐terminal flank and could also protein‐splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.
Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica
Authors: Eriksson Sofia , Lucchini Sacha , Thompson Arthur , Rhen Mikael , Hinton Jay C. D.
Published: March 2003

For intracellular pathogens such as Salmonellae, Mycobacteriae and Brucellae, infection requires adaptation to the intracellular environment of the phagocytic cell. The transition from extracellular to intravacuolar environment has been expected to involve a global modulation of bacterial gene expression, but the precise events have been difficult to determine. We now report the complete transcriptional profile of intracellular Salmonella enterica sv. Typhimurium following macrophage infection. During replication in murine macrophage‐like J774‐A.1 cells, 919 of 4451 S. Typhimurium genes showed significant changes in transcription. The expression profile identified alterations in numerous virulence and SOS response genes and revealed unexpected findings concerning the biology of the Salmonella–macrophage interaction. We observed that intracellular Salmonella are not starved for amino acids or iron (Fe2+), and that the intravacuolar environment is low in phosphate and magnesium but high in potassium. S. Typhimurium appears to be using the Entner–Douderoff pathway to use gluconate and related sugars as a carbon source within macrophages. Almost half the in vivo‐regulated genes were of unknown function, suggesting that intracellular growth involves novel macrophage‐associated functions. This is the first report that identifies the whole set of in vivo‐regulated genes for any bacterial pathogen during infection of mammalian cells.
Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome
Authors: Robinson Bridget A. , Welch Teresa L. , Smith Joseph D.
Published: March 2003

Plasmodium falciparum
‐infected erythrocytes sequester from blood circulation by binding host endothelium. A large family of variant proteins mediates cytoadherence and their binding specificity determines parasite sequestration patterns and potential for disease. The aim of the present study was to understand how binding properties are encoded into family members and to develop sequence algorithms for predicting binding. To accomplish these goals computational approaches and a binding assay were used to characterize adhesion across
Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins in the 3D7 parasite genome. We report that most family members encode the capacity to bind CD36 in the protein's semi‐conserved head structure and describe the sequence characteristics of a group of PfEMP1 proteins that do not. Structural and functional grouping of PfEMP1 proteins based upon head structure and additional domain architectural properties provide new insights into the protein family. These can be used to investigate the role of proteins in malaria pathogenesis and potentially tailor vaccines to recognize particular binding variants.
Demonstration of a sensory rhodopsin in eubacteria
Authors: Jung Kwang‐Hwan , Trivedi Vishwa D. , Spudich John L.
Published: March 2003

We report the first sensory rhodopsin observed in the eubacterial domain, a green light‐activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all‐trans retinal to form a pink pigment (λmax 543 nm) with a photochemical reaction cycle of 110 ms half‐life (pH 6.8, 18°C). Co‐expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane‐embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS‐PAGE and autofluorography of 3H‐labelled all‐trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix–helix interactions with membrane‐embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.
Multisite optical recording of excitability in the enteric nervous system
Authors: Neunlist  , Peters  , Schemann
Published: March 2003

A multisite optical recording technique consisting of an array of 464 photodiodes was used to measure dynamic changes in transmembrane potentials (Vm) of guinea‐pig and mouse enteric neurones stained with the voltage‐sensitive dye Di‐8‐ANEPPS. Optical recordings of Vm changes in enteric neurones which were evoked by depolarizing current pulses or synaptic activation mirrored the Vm changes measured intracellularly in the same neurone. Action potentials had fractional change in fluorescence of –0.09 ± 0.06% and their peak to peak noise level was 20 ± 14% of the action potential amplitude. Optical recordings after electrical stimulation of interganglionic nerve strands revealed slow EPSPs, nicotinergic supra‐ and subthreshold fast EPSPs as well as propagation of action potentials along interganglionic strands. Local application of acetylcholine onto a single ganglion induced reproducibly and dose dependently action potential discharge demonstrating the feasibility of neuropharmacological studies. The optical mapping made it possible to record action potentials simultaneously in a large number of neurones with high spatiotemporal resolution that is unattainable by conventional techniques. This technique presents a powerful tool to study excitability spread within enteric circuits and to assess differential activation of enteric populations in response to a number of stimuli which modulate neuronal activity directly or through synaptic mechanisms.
Slow transit constipation: a model of human gut dysmotility. Review of possible aetiologies
Authors: Knowles  , Martin
Published: March 2003

Slow transit constipation is a severe condition of gut dysmotility that predominantly affects young women and may result in surgical intervention. Current medical treatments for STC are often ineffective, and the outcome of surgery is unpredictable. STC was first described almost a century ago. Since this time, progress in improving therapy for this condition has been complicated by a lack of understanding of the aetiology, and great variation in the methods and criteria used for the study of patients with this debilitating disorder. It is difficult to find unequivocal data, and harder still to give a definitive picture of the cause or causes of STC. Here we consider the evidence for various aetiologies of STC, in the light of the physiological and pathological findings.
Neuronal control of the pyloric sphincter of the guinea‐pig
Authors: Yuan S. Y. , Costa M. , Brookes S. J. H.
Published: March 2003

The pyloric sphincter (PS) controls gastric emptying and prevents the reflux of duodenal content into the stomach. Neuronal pathways and reflexes controlling the guinea‐pig PS were physiologically investigated in isolated preparations. Simultaneous intracellular or extracellular and tension recordings from PS circular muscle with electrical and stretch stimulation were used. Electrical stimulation evoked an initial small contraction followed by a relaxation with a corresponding inhibitory junction potential (IJP) then a second large contraction with a corresponding excitatory junction potential (EJP). Hyoscine (1 μmol L–1) blocked the first contraction, and reduced the second contraction and EJP by 52.5% and 61%, respectively. These responses were further reduced by the NK2 antagonist, MEN10627 (1 μmol L–1), and the NK1 antagonist, SR140333 (1 μmol L–1). N‐nitro‐L‐arginine (100 μmol L–1) and apamin (0.5 μmol L–1) blocked the relaxation and the IJP. Duodenal electrical stimulation evoked an EJP, whereas antral stimulation evoked an IJP followed by a small EJP. All were blocked by hexamethonium (100 μmol L–1). Duodenal stretch evoked tetrodotoxin‐sensitive reflex contractions and membrane depolarization with action potentials in the PS. Thus, PS enteric motor neurones receive inputs from the duodenum and the stomach. There are stretch‐sensitive ascending excitatory reflex pathways from the duodenum to the PS.
Potentiation of motilin‐induced contraction by nitric oxide synthase inhibition in the isolated chicken gastrointestinal tract
Authors: KITAZAWA T. , ONODERA C. , TANEIKE T.
Published: March 2003

The present experiments were designed to determine whether or not endogenous nitric oxide (NO) modifies the contractile response to chicken motilin (ch‐MT) in the gastrointestinal (GI) tract (proventriculus and small intestine) of the chicken. ch‐MT (1 nmol L−1–1 μmol L−1) caused contractions of longitudinal muscle strips of the proventriculus through both myogenic and neurogenic (mostly cholinergic) mechanisms. On the other hand, ch‐MT (0.1 nmol L−1–100 nmol L−1) contracted the small intestine (duodenum, jejunum and ileum) only through a myogenic mechanism. L‐Nitroarginine methylester (L‐NAME) potentiated, and L‐arginine inhibited, the ch‐MT‐ induced contraction without affecting the responsiveness of acetylcholine (ACh) or 5‐hydroxytryptamine in the proventriculus. Electrical field stimulation (EFS)‐ and 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP)‐ induced contractions were also potentiated by L‐NAME. The potentiation by L‐NAME was prevented by L‐arginine but not by D‐arginine. However, in the presence of atropine or tetrodotoxin, neither L‐NAME nor L‐arginine modified the responses to ch‐MT and DMPP. In contrast to the proventriculus, L‐NAME and L‐arginine were both ineffective in modifying the ch‐MT‐induced contraction in the small intestine. These results indicate that NO synthase inhibition potentiates the contractile response of ch‐MT, EFS and DMPP in the chicken proventriculus through reduction of endogenous NO‐mediated presynaptic inhibition on neural ACh release. However, NOS inhibition did not modify the myogenic (direct) action of ch‐MT in gastric and intestinal smooth muscles of the chicken.
Effect of successive swallows on oesophageal motility of normal volunteers, patients with Chagas’ disease and patients with idiopathic achalasia
Authors: Dantas R. O.
Published: March 2003

Abstract  Most frequently, ten swallows of a 5‐mL bolus of water are performed during oesophageal manometry. Our hypothesis is that five swallows may produce the same results. We studied the oesophageal contraction parameters of 40 volunteers, 75 patients with Chagas’ disease and 14 patients with idiopathic achalasia. Motility was recorded at 5, 10 and 15 cm above the lower oesophageal sphincter. The subjects performed ten swallows of a 5‐mL bolus of water alternated with ten dry swallows with an interval of at least 30 s. We measured the amplitude, duration, peristaltic velocity, number of failed and number of simultaneous contractions of the initial five and final five dry and wet swallows. The comparison of dry and wet swallows showed the differences already known. The comparison of the parameters of the initial five swallows with those of the final five swallows showed no differences. Thus, when the initial five or the final five swallows were considered, there was no change in the conclusions reached by the comparison of patients and volunteers and of dry and wet swallows. We conclude that five swallows may be sufficient for the manometric examination of oesophageal parameters in Chagas’ disease and idiopathic achalasia.
Effects of position on oesophageal function: studies using combined manometry and multichannel intraluminal impedance
Authors: Tutuian R. , Elton J. P. , Castell D. O. , Gideon R. Matthew , Castell J. A. , Katz P. O.
Published: March 2003

Abstract Multichannel intraluminal impedance (MII) allows assessment of intraoesophageal bolus transit. In the supine position, bolus transit is produced almost exclusively by peristaltic contractions; in the upright position, gravity also contributes to bolus transit. MII and peristaltic pressures were measured in four positions (0, 30, 60 and 90°) using ten swallows (5 cc each) of both water and viscous liquid with body position determined by random choice. Tracings were analysed for total bolus transit time: time interval between bolus entry at 20 cm above and bolus exit at 5 cm above the lower oesophageal sphincter (LOS) and contraction amplitudes at 5 and 10 cm above the LOS. Statistical comparison of mean values of all four body positions was done using anova and Bonnferoni post‐test. Ten normal subjects (five females and five males, age 24–45 years) completed the study. At each body position, liquid material transited faster (P < 0.001) than viscous material. Both liquid and viscous materials transited at lower inclinations (0 and 30°) significantly slower than at higher inclinations (60 and 90°). There was an almost perfect inverse linear correlation between angle of inclination and bolus transit time for both liquid (r = −0.99) and viscous (r = −1.00) boluses (Spearman correlation r > 0.99 and P < 0.02 for both substances). Contraction amplitudes for liquid vs viscous material were not significantly different at a given degree of inclination. Mean distal oesophageal amplitude declined with increasing inclination. Combined MII–OM identifies and quantifies the effects of gravity on the dichotomy between specific pressures measured by OM and function assessed as transit measured by MII.
Analysis of the PTEN gene in human meningiomas
Authors: Peters  , Wellenreuther  , Rollbrocker  , Hayashi  , Meyer‐Puttlitz  , Duerr  , Lenartz  , Marsh  , Schramm  , Wiestler  , Parsons  , Eng  , Von Deimling
Published: March 2003

N. Peters, R. Wellenreuther, B. Rollbrocker, Y. Hayashi, B. Meyer‐Puttlitz, E‐M. Duerr, D. Lenartz, D.J. Marsh, J. Schramm, O.D. Wiestler, R. Parsons, C. Eng & A. von Deimling (1998) Neuropathology and Applied Neurobiology24, 3–8

Analysis of the PTEN gene in human meningiomas

Previous observations demonstrated that the neurofibromatosis type 2 gene (NF2) plays an important role in the pathogenesis of the transitional, fibroblastic and malignant variants of human meningiomas. No specific genes have been associated with the pathogenesis of meningothelial meningiomas and with the progression to anaplastic meningiomas. However, allelic losses on chromosomal arms 1p, 10q and 14q have been implicated in the process of malignant progression. Recently, PTEN (phosphatase and tensin homolog deleted on chromosome ten) also termed MMAC1 (mutated in multiple advanced cancers 1) or TEP1 (TGF—regulated and epithelial cell‐enriched phosphatase), emerged as a candidate gene on chromosome 10q23.3. Initial studies revealed mutations of PTEN in limited series of glioblastomas, breast, kidney and prostate carcinomas mainly as cell lines. In order to evaluate the involvement of PTEN in the development of meningiomas, we have analysed the entire coding sequence of the gene in a series of 55 meningiomas (WHO grade I), 10 atypical meningiomas (WHO grade II) and 10 anaplastic meningiomas (WHO grade III). No PTEN mutations were seen in the WHO grade I meningiomas. However, one of the anaplastic meningiomas carried a somatic mutation. In addition, all tumours were examined for the presence of homozygous deletions of PTEN but these were not detected in any of the meningiomas. Our data suggest that mutations in PTEN are not involved in the formation of low grade meningiomas, but may contribute to malignant progression in a fraction of anaplastic meningiomas.
Dynamics of microglial activation in the spinal cord after cerebral infarction are revealed by expression of MHC class II antigen
Authors: Schmitt  , Brook  , Buss  , Nacimiento  , Noth  , Kreutzberg
Published: March 2003

Microglial reactivity associated with induction of MHC class II (HLA‐DR) antigen is a sensitive indicator for pathological events in the CNS. To assess the response of glial cells after lesions of supraspinal descending tracts, HLA‐DR, CD68 and GFAP were studied immunohistochemically on spinal cord tissue of 5 patients who died after unilateral infarction of the middle cerebral artery territory, and 5 control cases. In patients who died shortly after a stroke (4–14 days) increased HLA‐DR‐immunoreactivity (HLA‐DR‐IR) could be observed in the intermediate grey matter and in the ventral horn. The CD68‐IR was much less intense. After longer survival times (5 weeks to 4 months), HLA‐DR‐IR in the grey matter was clearly lower than that observed in the spinal cord of short survival times, but very abundant in the dorsolateral funiculus, specifically within the corticospinal tract. In white matter areas, CD68‐IR was almost identical to the HLA‐DR‐IR. Within the grey matter, CD68‐IR was similar to the control tissue. A moderate increase of GFAP‐positive astrocytes could be seen only in the grey matter after longer survival times. It seems probable, that the dynamics of HLA‐DR‐positive microglia reflect the early phagocytosis of presynaptic terminals by microglia in target regions of descending fibre tracts. In the white matter, the removal of degenerating axons by phagocytosing microglia expressing HLA‐DR and CD68 antigens is a slower process which occurs over a period of months.
Ki‐67 immunolabelling index is a prognostic indicator in childhood posterior fossa ependymomas
Authors: Bennetto  , Foreman  , Harding  , Hayward  , Ironside  , Love  , Ellison
Published: March 2003

Conventional histological evaluation and subclassification of childhood ependymomas poorly predict their biological behaviour. The Ki‐67 labelling index (Ki‐67 LI), a measure of growth fraction, correlates with the biological behaviour of several neoplasms, and this retrospective study tested the hypothesis that Ki‐67 LI is a prognostic indicator in childhood posterior fossa ependymomas. Immunocytochemistry using an antibody to Ki‐67 was undertaken on 5 μm sections of formalin‐fixed, paraffin‐embedded tissue from 74 cases of childhood (age <16 years.) posterior fossa ependymoma. A Ki‐67 LI was established by counting the proportion of labelled nuclei in more than 1000 cells from several histological fields. Several clinical and histological variables (including Ki‐67 LI) potentially associated with survival were entered into univariate and multivariate analyses using a Cox proportional hazards model.Variables that showed a significant and independent association with survival were Ki‐67 LI (P<0.002), whether total surgical resection had been achieved according to operation records (P<0.03), and whether no adjuvant therapy had been given (P<0.01). Age, sex, and the presence of necrosis or microvascular proliferation did not correlate with survival. In our defined population of patients with ependymomas, Ki‐67 LI is a strong prognostic indicator. We recommend that Ki‐67 LI is used in the histological evaluation of childhood posterior fossa ependymomas during trials of novel adjunctive therapies.
Membranous expression of glucose transporter‐1 protein (GLUT‐1) in embryonal neoplasms of the central nervous system
Authors: Loda M. , Xu X. , Pession A. , Vortmeyer A. , Giangaspero F.
Published: March 2003

The human erythrocyte GLUT‐1 is a transmembrane protein which facilitates transport of glucose in the cell in an energy‐independent fashion. Neuroectodermal stem cells show strong membrane immunoreactivitry with this marker at early developmental stages in rodents. Membranous expression by undifferentiated neuroectodermal cells gradually decreases while GLUT‐1 becomes confined to the endothelial cells, when these acquire blood–brain barrier function. We thus sought to determine whether GLUT‐1 expression was limited to embryonal neoplasms of the central nervous system (CNS) which are presumably derived from developmentally arrested neuroectodermal stem cells. Archival material of 40 primary CNS neoplasms were examined for immunoreactivity with anti‐GLUT‐1. This included both non‐embryonal neoplasms (18 astrocytic tumours, one ependymoma and three oligodendroglioma) and embryonal neoplasms (12 cerebellar medulloblastomas, four supratentorial PNETs and two atypical teratoid/rhabdoid tumours (AT/RhT)). In addition, cell lines and nude mice xenografts derived from both undifferentiated and differentiated tumours were assessed for GLUT‐1 immunoreactivity by both immunohistochemistry and Western blotting. All embryonal tumours, MBs and PNET xenografts consistently showed GLUT‐1 membrane staining. Non‐embryonal neoplasms were negative except for vascular staining. Membrane protein fraction of embryonal tumours cell lines immunoreacted by immunoblot with GLUT‐1, whereas the glioblastoma cell line was negative. Expression of GLUT‐1 supports the stem cell nature of the cells of origin of MBs, supratentorial PNET and AT/RhTs. As a result, GLUT‐1 is a useful marker to define the embryonal nature of CNS neoplasms.
Grading of diffusely infiltrating astrocytomas by quantitative histopathology, cell proliferation and image cytometric DNA analysis
Authors: Sallinen P. K. , Sallinen S. ‐L. , Helén P. T. , Rantala I. S. , Rautiainen E. , Helin H. J. , Kalimo H. , Haapasalo H. K.
Published: March 2003

The aim of the study was to evaluate the applicability of quantitative histopathology as an aid for grading diffusely infiltrating astrocytomas. Primary astrocytomas were analysed for parameters (mean nuclear size, mitosis count, area fraction of endothelial cells and tumour necrosis, area fraction of nuclei, and Ki‐67 (MIB‐1) labelling index), which are closely related to the World Health Organization (WHO) 1979 and WHO 1993 grading criteria. All estimates correlated with the WHO histopathological grade and patient outcome. According to the receiver–operating characteristics curve, the presence of tumour necrosis and mitosis count (cut‐off at 3 mitoses/mm2 of neoplastic tissue) showed the best sensitivity and specificity in separating patients with different survival. The multivariate survival analyses confirmed this result. A decision‐tree model was constructed based on these two variables: twig I with less than 3 mitoses/mm2, twig II with equal or more than 3 mitoses/mm2 but no necrosis, and twig III with tumour necrosis. This model was found to be more strongly associated with survival than the WHO 1979 or WHO 1993 grading schemes. Low‐malignancy astrocytomas (WHO grade II or twig I tumours) could be further divided into two prognostic categories by the image cytometric DNA analysis. The results put an emphasis on astrocytoma grading on mitosis counts (grade II vs. III) and tumour necrosis (grade III vs. IV). To standardize the sampling for mitosis counting, it is suggested that a parallel Ki‐67 immunostaining be used for the identification of the most proliferative areas.
vCJD – predicting the future?
Authors: Hilton D. A.
Published: March 2003

The recent emergence of variant Creutzfeldt–Jakob disease (vCJD) in the UK, and demonstration that vCJD is caused by the same prion strain that causes bovine spongiform encephalopathy, have led to concerns about the possibility of a human epidemic. Although only 79 cases of vCJD have occurred to date, it is likely that hundreds of thousands of infected cattle entered the human food chain in the late 1980s and early 1990s, and the average incubation period of vCJD is unknown. Mathematical models have not yet been able to give useful predictions of future numbers of cases, and in the absence of a blood test for vCJD, current attempts to reduce uncertainties about future numbers of cases are based on the accumulation of PrPSc in lymphoreticular tissues. Extensive lymphoreticular PrPSc accumulation has been seen in all cases of symptomatic vCJD so far examined, and in one case 8 months prior to the onset of symptoms. Animal models of prion disease suggest that lymphoreticular involvement occurs early in the incubation period and reliably predicts future neurological disease. Based on these data, large scale anonymous studies looking for PrP accumulation in surgically removed tonsillectomy and appendicectomy specimens are underway. Examination of the first 3000 specimens has not revealed any positive samples, but at the moment the significance of negative findings is uncertain. It is anticipated that by the time these studies are complete more data will be available on how early PrP can be demonstrated in lymphoreticular tissue in vCJD, which together with the results from examination of further samples, will allow some comment as to the likelihood of a large human vCJD epidemic.
Decreased concentration of apolipoprotein E in the cerebrospinal fluid after acute brain injury
Authors: Kay A. D. , Nicoll J. A. R.
Published: March 2003

Introduction:  Possession of the apolipoprotein E (APOE) ε4 allele is associated with increased deposition of amyloid β‐protein in patients who die after traumatic brain injury (TBI) (Nicoll et al. Nat Medical 1995; 1: 135), and unfavourable outcome after TBI (Teasdale et al. Lancet 1997; 350: 1069) and subarachnoid haemorrhage (SAH) (Niskakangas et al. Stroke 2001; 32: 1181). Evidence from experimental brain injury studies confirm that apolipoprotein E (ApoE=protein, APOE=gene) plays an important role in the response of the brain to injury. We report findings from a study testing the hypothesis that ApoE is altered in the cerebrospinal fluid (CSF) of patients after acute brain injury, and these alterations are related to injury severity and clinical outcome.

Materials and methods:  ApoE concentration was determined, by enzyme linked immunosorbant assay, in the CSF of patients with SAH (N = 19), TBI (N = 13), and controls (N = 13) investigated for hydrocephalus or suspected shunt dysfunction. Injury severity was determined using the Glasgow Coma Score (GCS) and clinical outcome using the Glasgow Outcome Score (GOS).

Results:  The concentration of ApoE was significantly lower in the CSF of patients with acute brain injury (P < 0.001) compared with controls, and the magnitude of the reduction correlated with injury severity.

Conclusion:  The concentration of ApoE in the CSF decreases substantially after acute brain injury despite the likely leakage of plasma ApoE into the subarachnoid space. We speculate that ApoE is retained within the parenchyma of the brain where it may participate in the injury response.
Timing subdural haematoma
Authors: Mohamed S. , Rezaie P. , Al‐Sarraj S.
Published: March 2003

Introduction:  One specific technique by which subdural haematoma can be timed has been one of the most challenging problems faced by pathologists. Currently, the most documented technique used for timing trauma is the Perl's stain for haemosiderin indicative of two days time lag between trauma and death. Trauma leads to the activation of a group of cells known as macrophages and offers a possible new means of timing trauma. The aim of this project was to reach a sensible conclusion about timing subdural haematoma by using a variety of markers against macrophages.

Materials and methods:  Using dural tissue from 28 subjects diagnosed with subdural haematoma, a three step amplification protocol (DAKO, UK) was applied to 5–7 µm sections of the dura, with DAB as chromogen, for subsequent light microscopic analysis.

Results:  Both qualitative (using a simple scoring system) and quantitative analysis were carried out in this study. Quantitative analysis comprised image capture using a digital camera mounted on a Zeiss axioplan microscope and image analysis by calculation of the percent areas of immunoreactivity per section using Optimas v6.2.

Conclusions:  PGM 1 and MHC II are not definitive at dating subdural haematoma within the first 48 h period. The early expression of these antibodies makes them good diagnostic tools for dating trauma when combined with other methods such as the Perl's stain.
Differential effects of antioxidants on amyloid β‐ and H2O2‐induced cell death in the SHSY‐5Y neuronal cell‐line
Authors: Shukla C. , Plant L. D. , Henderson Z. , Bridges L. R. , Pearson H. A.
Published: March 2003

Introduction:  Amyloid β (Aβ) protein, a 38–42 amino acid peptide is the major component of senile plaques in Alzheimer's disease and has been suggested to exert its toxic effects via the direct and indirect production of reactive oxygen species.

Materials and methods:  We have investigated the role of reactive oxygen species in the toxic actions of Aβ on a human neuronal cell line SHSY‐5Y, using the MTT assay for cellular viability and comparing the toxicity of Aβ with that of H2O2.

Results:  The neurotoxic fragment of Aβ protein, Aβ25–35, had clear concentration‐ and time‐dependent toxic effects on SHSY‐5Y cells. The ability of antioxidants to reverse these toxic effects of 10 µm Aβ25–35 or 200 µm H2O2 were tested. Vitamin E (10–100 µm) did not affect either Aβ or H2O2. Trolox, a vitamin E analogue (5–200 µm) had protective effects on H2O2‐induced cell death but was ineffective in preventing the cell death induced by Aβ. Vitamin C (10–1000 µm) partially reversed H2O2‐induced toxicity but had no protective action when cells were exposed to Aβ.

Discussion:  These results indicate that the mechanisms by which Aβ and H2O2 exert their toxic effects are different and suggest that in this experimental system, formation of reactive oxygen species do not have major role in Aβ‐induced cell death.