bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein
Authors: Lindow Janet C. , Kuwano Masayoshi , Moriya Shigeki , Grossman Alan D.
Published: March 2003

The Bacillus subtilis structural maintenance of chromosomes (SMC) protein is a member of a large family of proteins involved in chromosome organization. We found that SMC is a moderately abundant protein (∼1000 dimers per cell). In vivo cross‐linking and immunoprecipitation assays revealed that SMC binds to many regions on the chromosome. Visualization of SMC in live cells using a fusion to the green fluorescent protein (GFP) and in fixed cells using immunofluorescence microscopy indicated that a portion of SMC localizes as discrete foci in positions similar to that of the DNA replication machinery (replisome). When visualized simultaneously, SMC and the replisome were often in similar regions of the cell but did not always co‐localize. Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins thought to interact with SMC. Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA.
A central regulator of morphological differentiation in the multicellular bacterium Streptomyces coelicolor
Authors: Nguyen Kien T. , Willey Joanne M. , Nguyen Liem D. , Nguyen Lieu T. , Viollier Patrick H. , Thompson Charles J.
Published: March 2003

In the multicellular bacterium Streptomyces coelicolor, functions of developmental (bald) genes are required for the biosynthesis of SapB, a hydrophobic peptidic morphogen that facilitates aerial hyphae formation. Here, we show that aerial hyphal growth and SapB biosynthesis could be activated independently from the normal developmental cascade by providing unprogrammed expression of functionally interactive genes within the ram cluster. ramC, ramS and ramR were essential for normal growth of aerial hyphae, and ramR, a response regulator gene, was a key activator of development. The ramR gene restored growth of aerial hyphae and SapB formation in all bald strains tested (albeit only weakly in the bldC mutant), many of which are characterized by physiological defects. Disruption of the ramR gene abolished SapB biosynthesis and severely delayed growth of aerial hyphae. Transcription of ramR was developmentally controlled, and RamR function in vivo depended on its putative phosphorylation site (D53). We identified and mapped RamR targets immediately upstream of the region encoding ramC and ramS, a putative operon. Overexpression of ramR in the wild‐type strain increased SapB levels and caused a distinctive wrinkled surface topology. Based on these results, we propose that phenotypes of bald mutations reflect an early stage in the Streptomyces developmental programme similar to the spo0 mutations in the unicellular bacterium Bacillus subtilis, and that RamR has analogies to Spo0A, the Bacillus response regulator that integrates physiological signals before triggering endospore formation.
Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs
Authors: Kresse Andreas U. , Dinesh Sriramulu D. , Larbig Karen , Römling Ute
Published: March 2003

Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS6100‐induced coupled insertion‐inversion mechanism. A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O‐antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.
The identification of circular extrachromosomal DNA in the nuclear genome ofTrypanosoma brucei
Authors: Alsford N. S. , Navarro M. , Jamnadass H. R. , Dunbar H. , Ackroyd M. , Murphy N. B. , Gull K. , Ersfeld K.
Published: March 2003

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such asLeishmaniaandTrypanosoma cruzi, but never inTrypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus ofTrypanosoma brucei. This novel type of DNA was termed NR‐element (NlaIIIrepeat element). In contrast to drug‐induced episomes in other kinetoplastids, theT. bruceiextrachromosomal NR‐element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR‐element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR‐element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG‐bearing chromosomes ofT. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events inT. brucei.
Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds
Authors: Madrid Martan P. , Di Pietro Antonio , Roncero M. Isabel G.
Published: March 2003

Chitin, a β‐1,4‐linked polysaccharide of N‐acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N‐acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin α‐tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild‐type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.
DNA binding properties of the HrmR protein of Nostoc punctiforme responsible for transcriptional regulation of genes involved in the differentiation of hormogonia
Authors: Campbell Elsie L. , Wong Francis C. Y. , Meeks John C.
Published: March 2003

Nostoc punctiforme
is an example of a filamentous cyanobacterium that is capable of differentiating non‐growing cells that constitute gliding filaments termed hormogonia. These gliding filaments serve in short distance dispersal and as infective units in establishing a symbiosis with plants, such as the bryophyte
Anthoceros punctatus
. Mutants of
N
.
punctiforme
exist which show elevated levels of initial infection of
A
.
punctatus
as a consequence of repeated cycles of hormogonium differentiation. Such mutations occur within the
hrmA
and
hrmU
genes. Further characterization of the
hrm
locus revealed several genes with an organizational and predicted protein sequence similarity to genes of heterotrophic bacteria that are involved in hexuronic acid metabolism. Genes in the
N. punctiforme
locus are transcribed in response to the presence of a plant extract containing hormogonium‐repressing factors. A predicted transcriptional repressor encoded in the locus, HrmR
,
was shown herein to be a specific DNA binding protein that regulates the transcription of its own gene and that of
hrmE
, a nearby gene. The ability of HrmR to bind DNA was abolished upon addition of either galacturonate or lysate from specifically induced
N
.
punctiforme
cells, implying that the
in vivo HrmR binding activity is modulated via an internal compound, most likely a sugar molecule.
A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor A3(2)
Authors: Grantcharova Nina , Ubhayasekera Wimal , Mowbray Sherry L. , McCormick Joseph R. , Flärdh Klas
Published: March 2003

Streptomyces coelicolor
A3(2) undergoes at least two kinds of cell division: vegetative septation leading to cross‐walls in the substrate mycelium; and developmentally regulated sporulation septation in aerial hyphae. By isolation and characterization of a non‐sporulating
ftsZ
mutant, we demonstrate a difference between the two types of septation. The
ftsZ17
(Spo) allele gave rise to a classical
white
phenotype. The mutant grew as well as the parent on plates, and formed apparently normal hyphal cross‐walls, although with a small reduction in frequency. In contrast, sporulation septation was almost completely abolished, resulting in a phenotype reminiscent of
whiH
and
ftsZΔ2p
mutants. The
ftsZ17
(Spo) allele was partially dominant and had no detectable effect on the cellular FtsZ content. As judged from both immunofluorescence microscopy of FtsZ and translational fusion of
ftsZ
to
egfp
, the mutation prevented correct temporal and spatial assembly of Z rings in sporulating hyphae. Homology modelling of
S. coelicolor
FtsZ indicated that the mutation, an A249T change in the C‐terminal domain, would be expected to alter the protein on the lateral face of FtsZ protofilaments. The results suggest that cytokinesis may be developmentally controlled at the level of Z‐ring assembly during sporulation of
S. coelicolor A3(2).
Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator
Authors: Pohl Ehmke , Haller Jon C. , Mijovilovich Ana , Meyer‐Klaucke Wolfram , Garman Elspeth , Vasil Michael L.
Published: March 2003

Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals. Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration. In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the ferric uptake regulator (Fur). Fur controls a wide variety of basic physiological processes including iron uptake systems and the expression of exotoxin A. Here, we present the first crystal structure of Fur from P. aeruginosa in complex with Zn2+ determined at a resolution of 1.8 Å. Furthermore, X‐ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution. The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.
Versatility of inner membrane protein biogenesis in Escherichia coli
Authors: Fröderberg Linda , Houben Edith , Samuelson James C. , Chen Minyong , Park Sei‐Kyoung , Phillips Gregory J. , Dalbey Ross , Luirink Joen , De Gier Jan‐Willem L.
Published: March 2003

To further our understanding of inner membrane protein (IMP) biogenesis in Escherichia coli, we have accomplished the widest in vivo IMP assembly screen so far. The biogenesis of a set of model IMPs covering most IMP structures possible has been studied in a variety of signal recognition particle (SRP), Sec and YidC mutant strains. We show that the assembly of the complete set of model IMPs is assisted (i.e. requires the aid of proteinaceous factors), and that the requirements for assembly of the model IMPs into the inner membrane differ significantly from each other. This indicates that IMP assembly is much more versatile than previously thought.
Functions of the CckA histidine kinase in Caulobacter cell cycle control
Authors: Jacobs Christine , Ausmees Nora , Cordwell Stuart J. , Shapiro Lucy , Laub Michael T.
Published: March 2003

The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. Because of its critical role, CtrA activity is temporally regulated by multiple mechanisms including phosphorylation and ClpXP‐dependent degradation of CtrA. The CckA histidine kinase is known to contribute to CtrA phosphorylation. We show here that genes differentially expressed in a ctrA temperature‐sensitive (ts) mutant are similarly affected in a cckA ts mutant, that the phosphorylation of CckA coincides temporally with CtrA phosphorylation during the cell cycle, and that CckA is essential for viability because it is required for CtrA phosphorylation. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. CckA also positively regulates CtrA activity by a mechanism that is independent of CtrA phosphorylation. CtrA is more stable in the presence of CckA than it is absence, suggesting that CckA may also be involved, directly or indirectly, in the regulation of CtrA proteolysis.
Luminal cross‐sectional area and tension–strain relation of the porcine bile duct
Authors: Duch B. U. , Petersen J. A. K. , Gregersen H.
Published: March 2003

The bile duct is a distensible tube serving to transport bile from the liver and gallbladder to the duodenum. The purpose of this study was to characterize the luminal cross‐sectional area (CSA) and tension–strain properties during distension of the normal isolated porcine common bile duct in vitro. An impedance planimetric system located inside a balloon was used. Eleven porcine bile ducts were examined in two locations, in the hepatic duct and in the common bile duct. The CSAs obtained in the common bile duct were significantly higher than those in the hepatic duct in the pressure range 0–8 kPa (P < 0.001). The circumferential wall tension (T) – strain (ɛ) relations for both locations fitted to the exponential equation T = a·eb·ɛ with determination coefficients of 0.97 ± 0.01. The a and b constants were not statistically different between the two locations indicating that the elastic properties did not differ. In conclusion, the luminal CSAs were larger in the common bile duct when compared to the hepatic duct and the tension–strain relations did not differ between the two segments of the bile duct.
Small intestinal dysmotility following abdominal irradiation in the rat small intestine
Authors: FRASER  , FRISBY  , BLACKSHAW  , SCHIRMER  , HOWARTH  , YEOH
Published: March 2003

Abdominal symptoms such as diarrhoea, abdominal cramps and vomiting are common during and after abdominal radiotherapy for gynaecological and pelvic malignancy. It has recently been recognized that small intestinal dysmotility may contribute to these symptoms but the underlying mechanisms are unclear in part because of the technical difficulties inherent in performing studies in irradiated small intestine. The aim of the current study was to evaluate small intestinal motor activity using perfused micromanometric techniques in 6–8‐cm segments of ileum during arterial perfusion with isotonic oxygenated fluorocarbon solution. Intestinal segments from six rats were studied 4 days after treatment with 10 Gy abdominal irradiation. Ileal segments from nine nonirradiated animals acted as controls. For each experiment the total number of pressure waves, high‐amplitude (>20 mmHg, long‐duration >6 sec) pressure waves, and long (>20 associated) bursts of pressure waves were determined.

Irradiation had no effect on the overall number of pressure waves, but increased high‐amplitude long‐duration (HALD) pressure waves (248 vs 7, P < 0.01). In control animals HALD waves were localized to a single recording site but after radiotherapy 74% of HALD waves were temporally associated with similar pressure waves in other manometric channels. Forty‐seven per cent of associated HALD waves migrated aborally. Retrograde migration of HALD waves was seen in five segments following irradiation. Irradiation abolished bursts of >20 pressure waves.

High‐amplitude contractions which migrate aborally are likely to contribute to diarrhoea after abdominal irradiation. The in vitro small animal model of radiation enteritis will permit interventions to provide further insights into the mediation of intestinal dysmotility.
Electrical mapping of the projections of intrinsic primary afferent neurones to the mucosa of the guinea‐pig small intestine
Authors: BERTRAND  , KUNZE  , BORNSTEIN  , FURNESS
Published: March 2003

The patterns of innervation of the mucosa by axons of individual primary afferent neurones with cell bodies in the myenteric plexus were studied by mapping sites from which electrical stimulation of the mucosa elicited action potentials (APs) in their cell bodies. Segments of guinea‐pig ileum were dissected to reveal the myenteric plexus over half of the intestinal circumference, leaving the mucosa intact over the other half. Intracellular recordings were taken from myenteric neurones located within 1 mm of the intact mucosa. Focal electrical stimuli were applied to the mucosa at multiple locations separated by about 1 mm. Neurones that responded had round or oval cell bodies with several long processes (Dogiel type II) and APs that had an inflection on the falling phase (AH‐neurones). Responses consisted of single APs or bursts of APs. Maps of the mucosal projections of 30 neurones were generated. The maximum distances from which individual neurones responded were 7 mm circumferential and 2 mm oral or anal to the cell body with a higher proportion of responses from the oral regions. The areas of intact mucosa calculated to be innervated ranged from 1 mm2 up to ≈15 mm2 (mean 3.9 mm2; median 2.5 mm2). It is estimated that the areas innervated would be two to three times larger under conditions where part of the mucosa is not removed. Some neurones also responded to a chemical or a mechanical stimulus applied to the mucosa within the electrically mapped area. It is concluded that intrinsic primary afferent neurones have overlapping receptive fields with 230–350 neurones innervating the same region of mucosa.
Activation of an adrenergic and vagally‐mediated NANC pathway in surgery‐induced fundic relaxation in the rat
Authors: Boeckxstaens  , Hirsch  , Kodde  , Moojen  , Blackshaw  , Tytgat  , Blommaart
Published: March 2003

The aim of this study was to pharmacologically characterize and investigate the possible contribution of adrenergic and nonadrenergic noncholinergic (NANC) pathways involved in the relaxation of the rat gastric fundus following abdominal surgery.

Using an intragastric balloon, the effect of skin incision (SI), laparotomy (LT) and manipulation of the small intestine followed by caecal resection (M + R) on fundic pressure was evaluated.

SI resulted in a brief relaxation of the gastric fundus abolished by guanethidine and blocked by hexamethonium and the combination of phentolamine, propranolol and atropine (PPA). LT induced a longer lasting relaxation which was abolished by guanethidine and hexamethonium. It was blocked by PPA and the combination of ganglionectomy and vagotomy, but unaffected by atropine, vagotomy or ganglionectomy. M + R induced a long‐lasting relaxation which was only partly blocked by guanethidine or PPA, illustrating an inhibitory NANC component. Vagotomy combined with guanethidine completely abolished the relaxation following M + R, whereas it was significantly blocked by hexamethonium and the combination of ganglionectomy with vagotomy.

These results indicate that SI, LT and M + R induce inhibition of fundic motility via an adrenergic mechanism. During M + R, an additional vagally mediated inhibitory NANC pathway is activated. Finally, we suggest that LT and M + R inhibit the gastric fundus via both a splanchnic and a vagal reflex pathway.
Prolonged stationary colonic motility recording in seven patients with severe constipation secondary to antidepressants
Authors: Leroi  , Lalaude  , Antonietti  , Touchais  , Ducrotte  , Menard  , Denis
Published: March 2003

The aim of this study was to determine whether the colonic motor profile of seven patients with constipation secondary to antidepressants differed from the motility of seven patients with idiopathic constipation and seven healthy volunteers. All constipated patients had very severe constipation. Colonic manometric recordings were performed for 24 h. The number of high amplitude propagating contractions (HAPC) was lower in the two groups of constipated patients than in controls. No HAPC were observed in 5/7 patients with constipation secondary to antidepressants and in 1/7 patients with idiopathic constipation. The overall area under the curve (AUC) in the left colon was lower in the two constipated patient groups than in controls. AUC increased after a 1000‐kcal standard meal given at noon in controls but not in the two groups of constipated patients. In conclusion, in patients with constipation secondary to antidepressants, the overall AUC was as poor as in patients with idiopathic constipation, and no colonic response to eating was observed. Moreover, the number of HAPC was more markedly decreased in patients with constipation secondary to antidepressants than in patients with idiopathic constipation.
The stomach’s response to unappetizing food: cephalic–vagal effects on gastric myoelectric activity
Authors: Stern R. M. , Jokerst M. D. , Levine M. E. , Koch K.L.
Published: March 2003

The aim of this investigation was to determine the effects of sham feeding food that was perceived as unappetizing on the cephalic–vagal reflex as measured by changes in gastric myoelectric activity. Thirty‐eight healthy human participants experienced one of two conditions: (i) an appetizing sham feeding condition in which participants chewed and expectorated two cooked frankfurters, and (ii) an unappetizing sham‐feeding condition in which participants chewed and expectorated two cold tofu frankfurters. All participants were asked to chew each mouth‐full of food 6–7 times and to be very careful not to swallow any of the food. Electrogastrograms (EGGs) were recorded for 10 min prior to, during, and for 15 min after sham feeding. A questionnaire was given to each participant after the procedure as a manipulation check and to assess food palatability. Results from the questionnaire showed, as expected, that the cooked frankfurters were significantly more appetizing than the cold tofu frankfurters (P < 0.01). In the group sham fed appetizing food, 3 cycles per minute (cpm) power increased during sham feeding, but the change was not significant; however, 3 cpm power decreased in the group sham fed unappetizing food. This difference was significant (P < 0.05). In conclusion, we have demonstrated that the cephalic–vagal reflex, as measured by power in the 3 cpm frequency region of the EGG, is sensitive to the subjective palatability of the food.
Biphasic alterations in gastrointestinal transit following endotoxaemia in mice
Authors: Ceregrzyn M. , Kamata T. , Yajima T. , Kuwahara A.
Published: March 2003

Lipopolysaccharide (LPS)‐induced alterations of gastrointestinal transit were studied in mice using activated charcoal. LPS (10 mg kg−1) induced biphasic alterations of intestinal transit. Increase (acceleration phase) and delay (lag phase) in gastrointestinal transit were observed at 90 and 480 min after LPS injection, respectively. LPS administration induced significant increases in tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and nitrate levels in blood serum with maximal levels observed at 1.5, 4, and 8 h after LPS administration, respectively. The effects of recombinant human lzactoferrin (rhLF) on LPS‐ induced alteration of gastrointestinal transit, and production of TNF‐α, IL‐1β and nitrate were also studied. Animals were pretreated with rhLF 24 hours before intraperitoneal administration of LPS. RhLF significantly increased gastrointestinal transit during the lag phase. In addition, rhLF decreased the level of TNF‐α in endotoxaemic animals. The levels of IL‐1β and nitrate were not significantly changed by rhLF. In conclusion, the effect of LPS on gastrointestinal transit is biphasic and the mechanism controlling the second phase most likely depends on TNF‐α production, while the first phase most likely does not depend on TNF‐α. On the other hand, it may be regulated by IL‐1β and nitric oxide production.
Bcl‐2 expressing T lymphocytes in multiple sclerosis lesions
Authors: Zettl  , Kuhlmann  , Brück
Published: March 2003

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. T lymphocytes play a major role in the pathogenesis of the disease. The exact mechanisms by which the inflammation is regulated in MS have not yet been defined. Studies in animal models of MS suggest that apoptosis of T cells is the main factor terminating inflammation. The process of apoptosis itself is regulated by a range of pro‐ and anti‐apoptotic proteins. The bcl‐2 gene family is an important member of these proteins. The present study investigated the expression of the anti‐apoptotic protein bcl‐2 in 11 chronic MS cases including five relapsing‐remitting and six chronic progressive MS patients. A total of 35 lesions containing all stages of demyelinating activity were studied. The number of CD3‐positive T cells and the absolute and relative numbers of T cells expressing bcl‐2 were determined by double immunocytochemistry. Bcl‐2 is expressed by T lymphocytes in MS plaques. Patients with chronic progressive MS have a higher proportion of bcl‐2 expressing T cells than patients with relapsing remitting disease. Highest numbers of bcl‐2‐positive T lymphocytes were found in remyelinating and demyelinated lesions, whereas active demyelinating lesions revealed lower numbers. These data indicate that cell‐death‐related proteins such as the anti‐apoptotic protein bcl‐2 are expressed in MS lesions and that they might have important effects on the regulation of elimination or persistence of inflammatory cells in the central nervous system.
Prion protein immunohistochemical staining in the brains of monkeys with transmissible spongiform encephalopathy
Authors: Baker  , Ridley  , Wells  , Ironside
Published: March 2003

Prion protein (PrP) immunohistochemical staining of the brains of common marmosets (Callithrix jacchus) with experimental transmissible spongiform encephalopathy is described. The monkeys (n=17) had been injected, intracerebrally, 17–49 months previously with homogenates of brain tissue taken post mortem from a cow with BSE (n=2 monkeys), a sheep with natural scrapie (n=2 monkeys), human cases of growth hormone related Creutzfeldt–Jakob disease (CJD) (n=2 monkeys), sporadic CJD (n=5 monkeys), or Gerstmann–Sträussler–Scheinker disease (GSS) (n=4 monkeys), or from monkeys with spongiform encephalopathy resulting from injection with brain tissue from these last two cases (n=1 monkey from each case). Only diffuse PrP‐staining was seen in monkeys injected with CJD‐material whereas more aggregated deposits of PrP were seen in monkeys injected with BSE‐, scrapie‐and GSS‐brain tissue. There were no patterns of staining specific to the brains injected with BSE‐material that could be used to identify the origin of that inoculum. BSE‐and scrapie‐injected monkey brains could be distinguished from each other because in BSE‐injected monkey brain the spongiform vacuolation was largely confined to subcortical structures whereas in scrapie‐injected monkey brain the spongiform vacuolation was also prominent in the neocortex. The patterns of PrP deposition differed markedly between those seen in monkey brains injected with BSE‐material or CJD‐material, but the patterns of PrP staining seen in monkey brains injected with BSE‐material were also seen in monkey brains injected with scrapie‐or GSS‐material. Overall there was a correlation between the length of the incubation period and the amount of aggregated PrP‐staining, but no correlation between the neuropathological picture and the clinical presentation of neurological signs.
Muscle fibre atrophy in critically ill patients is associated with the loss of myosin filaments and the presence of lysosomal enzymes and ubiquitin
Authors: Helliwell  , Wilkinson  , Griffiths  , McClelland  , Palmer  , Bone
Published: March 2003

Muscle wasting and weakness are common features of patients with critical illnesses, and may impair their recovery. This study examines whether cytoskeletal and contractile proteins are damaged, and which proteolytic mechanisms might be involved, in the muscle fibre atrophy or necrosis associated with the acute myopathy of critically ill patients. Ninety‐eight muscle biopsies were obtained by the conchotome method from 57 critically ill patients and examined morphometrically and by immunohistochemical labelling. Sequential biopsies showed a mean reduction in fibre cross‐sectional areas of 3–4% per day. More intense immunolabelling for desmin was seen in the smaller fibres of 52% of the biopsies, while immunolabelling for dystrophin, actin and myosin heavy chains was maintained. Myosin ATPase activity was weak in the smaller fibres in some biopsies, and electron microscopy showed the loss of myosin filaments in atrophic fibres. These changes suggest that loss of the filamentous structure of myosin, without degradation of the immunolabelled epitopes, leads to the collapse of the intermyofibrillar desmin network. Fibres with abnormal desmin labelling showed increased cathepsin B, lysozyme and ubiquitin immunolabelling. Nine cases showed increased immunolabelling for heat shock protein 72. The changes in desmin immunolabelling were more prevalent in patients with higher APACHE II scores on admission, but were not related to other clinical features. The results indicate that fibre atrophy is associated with myosin filament depolymerization and the presence of several proteolytic enzymes. In our study, these changes occurred in patients who were critically ill but who did not receive large doses of steroids or neuromuscular blocking agents.
No evidence for astrogliosis in brains of schizophrenic patients. A post‐mortem study
Authors: Falkai  , Honer  , David  , Bogerts  , Majtenyi  , Bayer
Published: March 2003

Schizophrenia is clinically and neuropsychologically characterized by severe cognitive and functional impairment suggesting the presence of a neurodegenerative process in the brains of affected individuals. A variety of neuroanatomical changes have been described such as loss and disorientation of neurons in grey and white matter and cortical atrophy. However, the neuropathological basis for schizophrenia is still unclear. In the present study we monitored the density of GFAP‐positive astrocytes in brains of 33 schizophrenic patients and 26 healthy controls. Both grey matter (entorhinal cortex and subiculum) and white matter (premotor cortex, subventricular zone of the third ventricle and next to inferior horn) structures were measured bilaterally. The overall finding was that there is no evidence for increased astrogliosis in brains of schizophrenic patients vs healthy controls. Therefore, degeneration is unlikely to be the main neuropathological mechanism in schizophrenic brains.
Tenascin‐R and C in multiple sclerosis lesions: relevance to extracellular matrix remodelling
Authors: Gutowski  , Newcombe  , Cuzner
Published: March 2003

In the present study the distribution of the inhibitory extracellular molecules tenascin‐R (TN‐R) and tenascin‐ C (TN‐C) was examined by immunocytochemistry during evolution of the multiple sclerosis (MS) lesion, in which astrogliosis is a prominent feature. Sections were cut from five control cases and from 22 blocks containing lesions representing different pathological stages in 18 cases of secondary progressive MS. Widespread expression of TN‐R was found in the normal human central nervous system (CNS), while that of TN‐C was in general restricted to white matter. In acute MS plaques however, there was a similar striking loss of both TN‐R and TN‐C up to the edge of the lesion, where the macrophage density is greatest, extending into the apparently normal white matter. In subacute lesions a TN‐C and/or TN‐R‐immunopositive reactive astrocyte subpopulation was prominent, reflecting synthesis of extracellular matrix molecules. Both tenascins were expressed throughout chronic MS plaques at levels similar to those seen in adjacent white matter. The loss of TN‐R and TN‐C in acute plaques is indicative of enzyme‐mediated breakdown of the matrix which may be a marker of blood–brain barrier breakdown and leucocyte extravasation. Subsequent production of tenascins by reactive astrocytes may result in glial scar formation impeding remyelination and axonal repair in MS lesions.
Microdysgenesis with abnormal cortical myelinated fibres in temporal lobe epilepsy: a histopathological study with calbindin D‐28‐K immunohistochemistry
Authors: Thom M. , Holton J. L. , D’Arrigo C. , Griffin B. , Beckett A. , Sisodiya S. , Alexiou D. , Sander J. W.
Published: March 2003

Microdysgenesis is a microscopic cortical malformation reported to occur with varying incidence in surgical lobectomies from patients with temporal lobe epilepsy (TLE). It may act as a substrate for the seizures. Four patients are reported with TLE, hippocampal sclerosis and cortical microdysgenesis which was also characterized by the presence of abnormal myelinated fibres running tangentially in the superficial cortical laminae and closely associated with abnormal clusters of neurones. Similar abnormal cortical fibres have been described in other malformations of cortical development including polymicrogyria and focal cortical dysplasia and it is therefore likely that these fibres represent part of the microdysgenetic malformation not hitherto reported. The possibility is discussed that they may also be of functional significance in terms of influencing local seizure propagation and the secondary cortical neuronal loss observed, predominantly affecting layer II. Studies of calbindin interneuronal populations showed preservation of these cells in the microdysgenetic cortex, when compared with non‐malformed temporal lobes, despite an overall reduction in cortical neuronal density. In addition, prominent numbers of neurogliaform calbindin‐positive nerve cells were observed in the microdysgenesis cases and the nature of these cells is speculated upon.
Immunocytochemical characterization of long‐term persistent immune activation in human brain after herpes simplex encephalitis
Authors: Lellouch‐Tubiana A. , Fohlen M. , Robain O. , Rozenberg F.
Published: March 2003

The clinical, virological and immunocytochemical features of three children who recovered from acute herpes simplex encephalitis (HSE) before the age of 2 years, and who developed secondary severe focal epilepsy after a symptom‐free period, leading to neurosurgery 3–10 years later are described. In one child, relapse of HSE occurred immediately after surgery. In all three patients, brain sample biopsies showed abundant CD3‐positive T lymphocytes with a majority of CD8 cells, and abundant activated macrophage‐microglial cells, a pattern similar to that found in acute HSE. Herpes simplex virus DNA was retrieved from the tissue biopsy in one case. The long‐term persistent cerebral inflammatory process observed after HSE differed from that observed in another chronic viral disease, subacute sclerosing panencephalitis. This inflammatory reaction may be a result either of low‐grade viral expression or self‐induced immune activation. The role of inflammation in triggering epilepsy remains hypothetical. Solving these issues should have major therapeutic implications. Herpes simplex virus DNA latency in brain may be the source of replicative HSE relapse.
Oedema and glial cell involvement in the aged mouse brain after permanent focal ischaemia
Authors: Fotheringham A. P. , Davies C. A. , Davies I.
Published: March 2003

This study examines the effect of age on oedema and brain swelling, and associated glial cell involvement on the size of the lesion in two models of permanent, focal cerebral ischaemia. Ischaemia was induced in male C57BL/Icrfat mice (4–6 and 26–31‐month‐old) by middle cerebral artery (MCA) occlusion using either electrocoagulation after craniotomy (MCA/craniotomy), or by an intraluminal filament through the carotid artery (MCA/icf). Twenty‐four hours after inducing ischaemia, brain swelling and lesion size were measured in young and aged mice, and cerebral oedema by wet/dry brain weights. Histopathology and immunocytochemistry were performed on a separate set of perfusion fixed brains. The MCA/icf technique produced a significantly larger lesion than MCA/craniotomy in both age groups. The percentage of water taken into the brain was significantly greater after MCA/icf, with aged mice showing the greatest increase. When lesion size was corrected for brain swelling there was no age‐related increase in the size of the lesion. The numbers of microglia and astroglia increased significantly in the parietal cortex of aged control animals, and there were qualitative differences in the glial response between the two stroke models. This study emphasizes the importance of age in models of permanent focal ischaemia, with oedema clearly being a significant factor. Differ‐ences in the responsiveness of the glial cell population with age may be of fundamental importance in the progress of ischaemic brain damage.
β‐Amyloid (Aβ)42(43), Aβ42, Aβ40 and apoE immunostaining of plaques in fatal head injury
Authors: Horsburgh K. , Cole G. M. , Yang F. , Savage M. J. , Greenberg B. D. , Gentleman S. M. , Graham D. I. , Nicoll J. A. R.
Published: March 2003

β‐Amyloid (Aβ) deposits are found in the brains of approximately one‐third of patients who die within days after a severe head injury; their presence correlating strongly with possession of an apolipoprotein E (apoE)‐ε4 allele. The aim of the study was to investigate the relationship between Aβ42, Aβ40 and apoE immunostaining of Aβ plaques in the cerebral cortex and the relevance of apoE genotype in 23 fatally head‐injured patients. These cases were known to have Aβ deposits from a previous study in which they were examined and semiquantified and related to apoE genotype. In the present study, the temporal cortex was probed using four different antibodies that recognize Aβ42(43), Aβ40 and an antibody to apoE. Aβ42(43)‐positive plaques were observed in all of the 23 cases and Aβ40 immunoreactivity in only 11 of the 23 cases. In addition, semiquantitative analysis showed that relatively fewer plaques were detected with anti‐Aβ40 than anti‐Aβ42(43). ApoE‐immunoreactive plaques were identified in 18 of the 23 cases. The number of plaques stained for apoE was relatively less than for Aβ42(43) but greater than for Aβ40. Furthermore, the density of Aβ plaques detected using either Aβ42(43), Aβ40 or apoE antibodies was associated with possession of apoE‐ε4 in an allele dose‐dependent manner. The results are consistent with Aβ42(43) as the initially deposited species in brain parenchyma and provide evidence that apoE is involved in the early stages of amyloid deposition. Further, the findings may be of relevance to the role of apoE genotype in influencing outcome after acute brain injury.
The relationship between αB‐crystallin and neurofibrillary tangles in Alzheimer's disease
Authors: Mao J. J. , Katayama S. , Watanabe C. , Harada Y. , Noda K. , Yamamura Y. , Nakamura S.
Published: March 2003

αB‐crystallin is known as a small heat shock protein with a cytoprotective function. This study was undertaken to investigate the relationship between αB‐crystallin and changes seen in Alzheimer's disease. The distribution and immunohistochemical characteristics of αB‐crystallin positive neurones in the cerebral cortices of 4 patients with Alzheimer's disease were examined. αB‐crystallin positive neurones were mainly distributed in the limbic and paralimbic regions, namely parahippocampal gyrus, fusiform gyrus, cingulate cortex, middle and superior frontal gyrus, and insular cortex, which corresponded to commonly affected regions in Alzheimer's disease. Moreover, such neurones were present predominantly in the III or V layer of the cerebral cortex. The number of αB‐crystallin positive neurones increased in parallel with the neuronal loss. Logistic regression analysis revealed a significant relation between the density of αB‐crystallin positive neurones and that of extracellular neurofibrillary tangles (NFTs), with a correlation coefficient (r) of 0.57 and P < 0.0001 in 14 regions of the cerebral cortex. In contrast, the relation was not statistically significant between the density of αB‐crystallin positive neurones and that of classical senile plaques, diffuse plaques or intracellular NFTs. Modified Gallyas–Braak (GB) staining on αB‐crystallin positive neurone demonstrated several patterns of the structures: faint GB positive structures in the swollen perikaryon with straight neurites, fine granules compressed and contorted into fuzzy bundles, intensely GB positive filamentous structures together with fine granules and very intensely GB positive ring‐like NFTs in a swollen perikaryon with curved neurites. In positive neurones, the density of ring‐like NFTs correlated with that of atrophic perikaryon, or bent neurites and a decrease in the immuoreactivity of αB‐crystallin. These data suggest that a close relationship exists between the appearance of αB‐crystallin in neurones, extracellular NFTs, and neurofibrillary formation in αB‐crystallin positive neurones in Alzheimer brain.
Quantitative electroencephalography spectral analysis and topographic mapping in a rat model of middle cerebral artery occlusion
Authors: Lu X.‐C. M. , Williams A. J. , Tortella F. C.
Published: March 2003

Electroencephalography (EEG) has a long history in clinical evaluations of cerebrovascular disease. Distinct EEG abnormalities, such as increased slow delta activity, voltage depression and epileptiform discharge, have been identified in stroke patients. However, preclinical use of EEG analysis of cerebral ischaemia is less documented. We report a new rat model of EEG topographic mapping during permanent and transient middle cerebral artery occlusion. Ten EEG electrodes were implanted on the rat skull, symmetrically covering the cortical regions of two hemispheres. Monopolar EEG recordings were acquired from each animal at multiple time points during the initial 24 h, and again once daily for 7 days. Traditional EEG examinations, quantitative EEG (qEEG) spectral analysis and topographic EEG mapping were employed for comprehensive data analyses. Several distinct spatiotemporal EEG abnormalities were identified in the ischaemic rat brain. In the ipsilateral hemisphere, pronounced increase in delta activity was observed in each recorded area within 24 h of injury. While sharp waves and spike complexes dominated the parietal region, a nearly isoelectric EEG state was seen in the temporal region. After 48 h, spontaneous, albeit incomplete, recovery of EEG activities developed in all rats. Reperfusion appeared to promote delta and alpha recovery more efficiently. The contralateral EEG changes were also recorded in two phases: an acute moderate increase in delta activities with intermittent rhythmic activities, followed by a delayed and significant increase in beta activities across the hemisphere. The similarities of rat qEEG profiles identified in this study to that of stroke patients and the application of topographic mapping broaden our research technology for preclinical experimental studies of brain injury.
What is the basis of transmissible spongiform encephalopathy induced neurodegeneration and can it be repaired?
Authors: Fraser J. R.
Published: March 2003

Once an animal becomes infected with a prion disease, or transmissible spongiform encephalopathy (TSE), the progression of infection is relentless and inevitably fatal, although often with such prolonged incubation periods that an alternative cause of death can intervene. Infection has been compared to ‘setting a clock’ which then runs inexorably as the disease spreads, usually through the lymphoreticular system and then via peripheral nerves to the central nervous system (CNS), although the mechanism controlling the protracted progression is not known. Clinical disease develops as characteristic degenerative changes in the CNS progress, but the molecular basis for this pathology is not clear, particularly the relationship between the deposition of abnormal PrP and neuronal dysfunction. Recent research has identified several means of slowing (if not stopping) the clock when infection has not yet reached the CNS; although the potential for later stage therapies seems limited, neuroprotective strategies which have been shown to be effective in other neurodegenerative conditions may also ameliorate TSE induced CNS pathology. This review focuses on our current knowledge of the key events following infection of the CNS and the opportunities for intervention once the CNS has become infected.
Transforming growth factor β1, the dominant cytokine in murine prion disease: influence on inflammatory cytokine synthesis and alteration of vascular extracellular matrix
Authors: Cunningham C. , Boche D. , Perry V. H.
Published: March 2003

Previous studies from our laboratory have shown the ME7 model of murine scrapie to be accompanied by an atypical inflammatory response that is characterized by marked astroglial and microglial activation but also by the lack of significant expression of the pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐6. The aim of this study was to determine whether, in the absence of IL‐1β and IL‐6, tumour necrosis factor (TNF)‐α may play an equivalent pro‐inflammatory role, or if an anti‐inflammatory cytokine profile dominates. We have used competitive polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) to determine the levels of TNF‐α, IL‐10 and transforming growth factor (TGF)‐β1 in the ME7 model, using their expression in lipopolysaccharide (LPS)‐induced acute inflammation as a positive control. Levels of mRNA were elevated for all three cytokines during acute inflammation, while TGF‐β1 mRNA alone was significantly elevated in ME7‐injected brains. Similarly, by ELISA, we detected elevated IL‐10, TNF‐α and TGF‐β1 in LPS‐injected animals but only significant elevation of TGF‐β1 in ME7‐injected animals. An increase in laminin and collagen IV deposition around blood vessels was also observed and is consistent with up‐regulation by active TGF‐β1. These findings suggest that TGF‐β1 may play a central role in maintenance of an atypical microglial phenotype and may also be involved in vascular and extracellular matrix changes.