bluuebunny/crossref_metadata_2025_split huggingface dataset:
The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo‐ and heterodimers in vivo
Authors: Mamnun Yasmine M. , Pandjaitan Rudy , Mahé Yannick , Delahodde Agnés , Kuchler Karl 
Published: March 2003

SummaryThe transcription factors Pdr1p and Pdr3p from Saccharomyces cerevisiae mediate pleiotropic drug resistance (PDR) by controlling expression of ATP‐binding cassette (ABC) transporters such as Pdr5p, Snq2p and Yor1p. Previous in vitro studies demonstrated that Pdr1p and Pdr3p recognize so‐called pleiotropic drug resistance elements (PDREs) in the promoters of target genes. In this study, we show that both Pdr1p and Pdr3p are phosphoproteins; Pdr3p isoforms migrate as two bands in gel electrophoresis, reflecting two distinct phosphorylation states. Most importantly, native co‐immunoprecipitation experiments, using functional epitope‐tagged Pdr1p/Pdr3p variants, demonstrate that Pdr1p and Pdr3p can form both homo‐ and heterodimers in vivo. Furthermore, in vivo footprinting of PDRE‐containing promoters demonstrate that Pdr1p/Pdr3p constitutively occupy both perfect and degenerate PDREs in vivo. Thus, in addition to interaction with other regulators, differential dimerization provides a plausible explanation for the observation that Pdr3p and Pdr1p can both positively and negatively control PDR promoters with different combinations of perfect and degenerate PDREs.
Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina
Authors: Pinan‐Lucarré Bérangère , Paoletti Mathieu , Dementhon Karine , Coulary‐Salin Bénédicte , Clavé Corinne 
Published: March 2003

SummaryIn filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi‐6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi‐7. We have used a GFP–IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi‐7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.
From pre‐cells to Eukarya – a tale of two lipids
Authors: Wächtershäuser G. 
Published: March 2003

SummaryA mechanistic hypothesis for the origin of the three domains of life is proposed. A population of evolving pre‐cells is suggested to have had a membrane of a racemate of chiral lipids that continuously underwent spontaneous symmetry breaking by spatial phase segregation into two enantiomerically enriched membrane domains. By frequent pre‐cell fusions and fissions these membrane domains became partitioned between two pre‐cell subpopulations having predominantly one lipid enantiomer or the other. The origin of the Bacteria and Archaea is explained by divergence of first a population of proto‐bacteria and later a population of proto‐archaea from the evolving pre‐cells, each by the emergence of an enantio‐selective lipid biosynthesis within the corresponding pre‐cell subtype. The origin of the Eukarya is explained by symbiosis between a population of Bacteria and a subpopulation of pre‐cells with a predominance of the bacteria‐type lipid enantiomers.
Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine‐rich sites
Authors: Hendrixson D. R. , Qiu J. , Shewry S. C. , Fink D. L. , Petty S. , Baker E. N. , Plaut A. G. , St. Geme J. W. 
Published: March 2003

SummaryLactoferrin is a member of the lactotransferrin family of non‐haem, iron‐binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N‐terminal lobe of the protein, adjacent to a 12–15 Å cleft that separates the N‐lobe and the C‐lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine‐rich region defined by amino acids 1379–1386 (RRSRRSVR) and digests Hap at an arginine‐rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine‐rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine‐rich sequences in a variety of microbial virulence proteins, contributing to its long‐recognized antimicrobial properties.
EspH, a new cytoskeleton‐modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli
Authors: Tu Xuanlin , Nisan Israel , Yona Chen , Hanski Emanuel , Rosenshine Ilan 
Published: March 2003

SummaryEnteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42‐dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.
Identification of a novel Gsp‐related pathway required for secretion of the manganese‐oxidizing factor of Pseudomonas putida strain GB‐1
Authors: De Vrind Johannes , De Groot Arjan , Brouwers Geert Jan , Tommassen Jan , De Vrind‐de Jong Elisabeth 
Published: March 2003

SummaryThe manganese‐oxidizing factor of Pseudomonas putida strain GB‐1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two‐gene operon that includes an xcpS homologue (designated xcmS). No other xcp‐like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB‐1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin‐related protein. This xcmX is clustered with two other xcpT‐related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well‐described ‘regular’xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp‐related system for the transport of manganese‐oxidizing enzymes to the cell surface.
Interaction of the sensor module of Mycobacterium tuberculosis H37Rv KdpD with members of the Lpr family
Authors: Steyn Adrie J. C. , Joseph Joan , Bloom Barry R. 
Published: March 2003

SummaryThe genetic and biochemical mechanisms by which Mycobacterium tuberculosis senses and responds to the complex environment that it encounters during infection and persistence within the host remain unknown. In a number of bacterial species, the Kdp signal transduction pathway appears to be the primary response to environmental osmotic stress, which is primarily mediated by K+ concentration in bacteria. We show that kdp encodes for components of a mycobacterial signalling pathway by demonstrating the K+ dependence of kdpFABC expression in both M. tuberculosis H37Rv and Mycobacterium smegmatis. To identify proteins of M. tuberculosis that participate in this signalling pathway, we used the N‐terminal sensing module of the histidine kinase KdpD as bait in a yeast two‐hybrid screen. We show that the sensing domain of KdpD interacts specifically with two membrane lipoproteins, LprJ (Rv1690) and LprF (Rv1368). Overexpression of lprF and lprJ alleles in mycobacterial kdpF–lacZ reporter strains enabled us to identify alleles that modulate kdpFABC expression. By exploiting the yeast three‐hybrid system, we have found that the histidine kinase domain of KdpD forms ternary complexes with LprF and LprJ and the sensing module of KdpD. Our results establish a role for membrane proteins in the Kdp signalling pathway and suggest that LprF and LprJ function as accessory or ligand‐binding proteins that communicate directly with the sensing domain of KdpD to modulate kdp expression.
Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae
Authors: Giots Frank , Donaton Monica C. V. , Thevelein Johan M. 
Published: March 2003

SummaryYeast cells starved for inorganic phosphate on a glucose‐containing medium arrest growth and enter the resting phase G0. We show that re‐addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE‐controlled genes and induction of ribosomal protein genes. Phosphate‐induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G‐protein coupled receptor Gpr1 and by the glucose‐phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport‐independent receptor. The non‐metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate‐starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long‐term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose‐dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.
Clinical isolates of Enterococcus faecium exhibit strain‐specific collagen binding mediated by Acm, a new member of the MSCRAMM family
Authors: Nallapareddy Sreedhar R. , Weinstock George M. , Murray Barbara E. 
Published: March 2003

SummaryA collagen‐binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen‐binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin‐resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI‐binding vancomycin‐resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non‐CI‐binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration‐dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm‐specific IgGs purified from anti‐recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen‐binding clinical isolates of E. faecium tested, but in none of the strains with a non‐functional pseudo acm gene. Introduction of a functional acm gene into two non‐CI‐binding natural acm mutant strains conferred a CI‐binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection‐derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.
Possible role of nitric oxide in postoperative ileus: a comparative study
Authors: Moojen  , Van Gulik T. M. , Hoek  , Gouma  , Tytgat  , Boeckxstaens  
Published: March 2003

In the present study, the possible involvement of nitric oxide (NO) in the pathogenesis of postoperative ileus was investigated indirectly by measuring nitrate, a stabile metabolite of NO.Plasma levels and 24‐h urinary excretion of nitrate and nitrite were determined in the peri‐operative period in three different groups of patients undergoing surgery: group 1 (LT, n=11) underwent a laparotomy, group 2 (LS, n=12) underwent a laparoscopic procedure, whereas group 3 underwent an extra‐abdominal procedure (EA, n=9).Duration of postoperative ileus was assessed clinically using first occurrence of flatus and defaecation as the end of the period of ileus.Postoperative ileus lasted significantly longer in the LT group (first flatus after 3.0 [3.0–4.0] days) compared with the LS (1.0 [1.0–2.0] days) and EA (1.0 [1.0–3.0] days) groups. Urinary nitrate excretion increased significantly in the LT and EA groups during the first 24 h after surgery (from 797.0 [214.0–810.0] and 551.5 [438.3–1215.8] to 2079.0 [889.0–4644.0] and 1102.5 [315.3–1238.0] μmol/24 h, median [IQR]), but normalized before the end of postoperative ileus. Plasma levels of nitrate were unchanged after surgery, whereas CRP levels were significantly increased in all groups (LT > LS=EA).In the first 24 h following surgery, urinary nitrate excretion is increased, suggesting increased endogeneous synthesis of NO postoperatively. As no correlation was found between urinary nitrate excretion and duration of postoperative ileus, we conclude that assessment of nitrate has no value in predicting clinical outcome after surgery.
Influences of pelvic floor structures and sacral innervation on the response to distension of the cat rectum
Authors: Mathis C. , Schikowski A. , Thewißen M. , Ross H‐G. , Crowell M. D. , Enck P. 
Published: March 2003

Abstract

The contributions to the rectal response to distension of the pelvic floor structures surrounding the rectum and of the sacral spinal innervation have never been studied. Using paralysed intercollicularly decerebrate, anaesthesia‐free cats, we studied pressure–volume relationships during slow ramp distensions of the rectum. Results obtained from animals with intact pelvic cavities were compared with those following mobilization of the rectum from the pelvic floor musculature. To assess the influences of spinal outflow and afferent input, rectal pressure–volume relationships were measured in the mobilized rectum following bilateral sequential transection of the spinal roots S1 to S3, first dorsal, then ventral. Isolation of the rectum from the pelvic floor structures resulted in a decrease in balloon volume in the lower range of distension pressure but did not affect volumes at higher pressures. The only afferent effect was seen after sectioning of dorsal roots S1, which resulted in a decrease in balloon volume. The only efferent effect was seen after sectioning of ventral roots S3, which decreased balloon volume further. In conclusion, the rectal response to distension depends on the properties of the rectal wall. It may be influenced by somatic inputs, inputs from the myenteric nervous plexus, and from the parasympathetic and sympathetic nervous systems. Afferent inputs and spinal autonomic reflexes may decrease the tone of the rectal musculature during distension.
Sensory–motor responses to volume‐controlled duodenal distension
Authors: Gao C. , Petersen P. , Liu W. , Arendt‐Nielsen L. , Drewes A. M. , Gregersen H. 
Published: March 2003

Abstract
Visceral perception and secondary peristalsis evoked by distension of the duodenum were studied in 10 healthy volunteers. An impedance planimetric probe for cross‐sectional area (CSA) measurements inside a balloon and with three pressure channels was used. Balloon distensions were performed in the fed state with or without the administration of the antimuscarinic drug butylscopolamine. A modified questionnaire was used to assess the nonpainful and painful sensations. The total tension (Ttotal) and the passive tension (Tpassive) were determined from the distensions without and with the administration of butylscopolamine, respectively. The active tension (Tactive) was Ttotal – Tpassive. The stepwise balloon distensions induced the first sensation at a volume of 33 ± 3 mL. After administration of butylscopolamine the first sensation appeared at 42 ± 1 mL. The perception score (PS) revealed an approximately linear increase as function of volume, CSA, pressure and tension after the first sensation. Butylscopolamine resulted in significant changes in PS score as function of volume, CSA and strain, but not as a function of pressure and tension. The frequency of the secondary peristalsis increased to the highest value (8.2 ± 0.8 contractions min−1) at a volume of 21 mL. Butylscopolamine almost abolished the distension‐evoked motility. Ttotaland Tpassiveincreased nonlinearly as a function of volume, whereas Tactiveincreased up to a distension volume of 33 mL and then decreased at higher volumes. Hence, the conventional length–tension diagrams as known from studies of smooth muscle strips in vitro can be reproduced in the human duodenum in vivo. This new way of studying intestinal sensation and motility may prove to have both basic and clinical importance as both passive tissue structures and the sensorimotor function are tested.
Association of APOE polymorphisms and pathological features in traumatic brain injury
Authors: Smith C. , Graham D. I. , Murray L. , Nicoll J. A. R. 
Published: March 2003

Introduction:  Previous studies have found that polymorphisms of the apolipoprotein E gene (APOE) influences the outcome from head injury, with possession of APOE ε4 being associated with an unfavourable outcome (Teasdale et al. Lancet 1997; 350: 1069–1071). In this study we correlated the pathological features of traumatic brain injury with the presence or absence of APOE ε4.Materials and methods:  During the 13‐year period 1987–99, 238 brains were examined from cases of traumatic brain injury (TBI) in the Department of Neuropathology, Glasgow. APOE genotype was determined by PCR. For each case a number of pathological features were recorded ‘blind’ to the APOE ε4 status.Results:  Of the 238 cases examined, 83 (35%) had the APOE ε4 allele, while 155 (65%) did not. Possession of APOE ε4 was associated with a greater incidence of severe hypoxic brain damage (ε4+ 54%, ε4– 42%) and with a greater incidence of moderate or severe contusion index (ε4+ 42%, ε4– 30%). No significant differences were noted between the other pathological features examined.Conclusions:  Possession of APOE ε4 is associated with a greater incidence of moderate/severe contusional injury and severe hypoxic brain damage in fatal cases of TBI. This may be relevant to the relatively poor outcome from TBI in patients with an APOE ε4 allele identified in clinical studies.
Muscle fibrillin deficiency associated with Marfan's syndrome myopathy
Authors: Behan W. M. H. , Longman C. , Petty R. K. H. , Boxer M. , Foskett P. , Harriman D. G. F. 
Published: March 2003

Introduction:  Marfan's syndrome (MFS) is the commonest inherited disorder of connective tissue. It is defined by major features in the cardiovascular, skeletal and ocular systems, and minor features in the integument and lungs. Patients who fulfil the diagnostic criteria have a mutation in the fibrillin (FBN1) gene, which encodes a major glycoprotein in the microfibrillar system, including in perimysium and endomysium. Muscle involvement, although in the original description, has not been mentioned at all recently.Materials and methods:  We studied a family with MFS in whom myopathy led to respiratory failure. Genetic analyses on venous blood and immunohistochemical studies of muscle biopsies were carried out.Results:  Single stranded conformational polymorphism screening of all exons of the FBN1 gene revealed a C4621 T base change in exon 37. This was predicted to result in the production of a truncated form of FBN1. Immunohistochemical studies showed conspicuous abnormalities in endomysial and perimysial fibrillin.Conclusion:  This is the first demonstration of fibrillin deficiency in muscle in MFS and its implications for biomechanical function are discussed.