bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetics of florfenicol in North American elk (Cervus elaphus)
Authors: Alcorn J. , Dowling P. , Woodbury M. , Killeen R.
Published: October 2004

Florfenicol pharmacokinetics after administration of a single subcutaneous (s.c.) dose of 40 mg/kg of body weight in adult elk (Cervus elaphus) was investigated. Serum florfenicol concentrations were determined by a sensitive high‐performance liquid chromatographic method with limit of quantification of 0.03 μg/mL. Florfenicol pharmacokinetic parameters in elk were estimated using a noncompartmental approach. After a single s.c. injection, florfenicol concentrations remained above 1 μg/mL for approximately 36 h and above 0.5 μg/mL for approximately 72 h. Following s.c. injection, florfenicol was absorbed rapidly with a mean maximum concentration (Cmax) of 3.7 μg/mL achieved at 4.2 h (Tmax). The Cmax value in elk is similar to values reported in cattle at the same dose, suggesting that the 40 mg/kg s.c. dose achieves therapeutic concentrations in elk. A mean elimination half‐life (t1/2) of 44 h is shorter than that reported in cattle. The more rapid elimination half‐life in elk suggests that elk may require a multiple dose regimen for therapeutic success with s.c. Nuflor®. We recommend s.c. Nuflor® be administered subcutaneously to elk every 24 h at a dose level of 40 mg/kg.
Therapeutic implications of the MDR‐1 gene
Authors: Mealey K. L.
Published: October 2004

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. P‐glycoprotein (P‐gp), the product of the MDR1 (ABCB1) gene, is among the most well‐characterized drug transporters, particularly in veterinary medicine. A number of clinically relevant, structurally and functionally unrelated drugs are substrates for P‐gp. P‐gp is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, P‐gp limits oral absorption and central nervous system entry of many substrate drugs. A number of MDR1 polymorphisms have been described in human patients, some of which result in altered drug pharmacokinetics and susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and others. An MDR1 polymorphism in herding breed dogs, including collies and Australian shepherds, has been demonstrated to be the cause of ivermectin sensitivity in these breeds. Recent evidence suggests that this polymorphism, a 4‐bp deletion mutation, results in increased susceptibility to the toxicity of several drugs in addition to ivermectin. Furthermore, data in rodent models suggest that P‐gp may play an important role in regulating the hypothalamic–pituitary–adrenal axis.
Angiotensin‐converting enzyme inhibitors in the therapy of renal diseases
Authors: Lefebvre H. P. , Toutain P. L.
Published: October 2004

Renal diseases, especially chronic renal failure (CRF), are common in canine and feline medicine. The renin‐angiotensin‐aldosterone system (RAAS) plays a pivotal role in these conditions in the development of renal lesions and the progression of kidney dysfunction. Angiotensin‐converting enzyme inhibitors (ACEI) are currently considered as the most efficient agents in therapeutic strategies. The benefit of an ACEI treatment can be explained by at least three mechanisms: ACEI limit systemic and glomerular capillary hypertension, have an antiproteinuric effect, and retard the development of glomerulosclerosis and tubulointerstitial lesions. These effects have been studied in dogs and cats, and there is now some evidence to support the recommendation of ACEI therapy in dogs and cats with CRF. Nevertheless the prescription of ACEI in such patients should take into account the potential influence of renal impairment on ACEI disposition, and adverse effects on the renal function itself (especially hypotension and acute reductions in glomerular filtration rate). The risk of drug interaction with diuretics, nonsteroidal anti‐inflammatory drugs and anesthetics, should not be overestimated. Furthermore, hypotension may occur in patients on a low sodium diet.
The rate of fertilization in male mating tactics of the polygynous grey seal
Authors: LIDGARD D. C. , BONESS D. J. , BOWEN W. D. , McMILLAN J. I. , FLEISCHER R. C.
Published: October 2004

Studies using molecular markers have shown that some grey seal males may be gaining success through exhibiting alternative mating tactics. We estimated the probability of fertilization success of grey seal males exhibiting the primary tactic of female defence and one alternative tactic of mating with departing females on Sable Island, Nova Scotia, Canada, during the breeding seasons of 1997–2002. Although the fertilization rate of the primary tactic (27–43%) was greater than that of the alternative tactic (10–12%), these low rates indicate the potential fitness value of alternative mating tactics in this size‐dimorphic pinniped species.
Geological barriers and restricted gene flow in the holarctic skipperHesperia comma(Hesperiidae)
Authors: FORISTER M. L. , FORDYCE J. A. , SHAPIRO A. M.
Published: October 2004

Patterns of genetic variation within a species may be a consequence of historical factors, such as past fragmentation, as well as current barriers to gene flow. Using sequence data from the mitochondrial cytochrome oxidase subunit II region (COII) and the nuclear genewingless, we conducted a phylogeographical study of the holarctic skipperHesperia commato elucidate patterns of genetic diversity and to infer historical and contemporary processes maintaining genetic variation. One hundred and fifty‐one individuals were sampled from throughout North America and Eurasia, focusing on California and adjacent regions in the western United States where morphological diversity is highest compared to the rest of the range. Analyses of sequence data obtained from both genes revealed a well‐supported division between the Old and New World. Within western North America,winglessshows little geographical structure, while a hierarchical analysis of genetic diversity of COII sequences indicates three major clades: a western clade in Oregon and Northern California, an eastern clade including the Great Basin, Rocky Mountains and British Columbia, and a third clade in southern California. The Sierra Nevada and the Transverse Ranges appear to be the major barriers to gene flow forH. commain the western United States. Relatively reduced haplotype diversity in Eurasia compared to North America suggests that populations on the two continents have been affected by different historical processes.
A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa
Authors: Galyov Edouard E. , Wood Michael W. , Rosqvist Roland , Mullan Paul B. , Watson Patricia R. , Hedges Samantha , Wallis Tim S.
Published: October 2004

Enteritis induced by non‐typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella‐induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip‐dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.
Bacterial cell division and the septal ring
Authors: Weiss David S.
Published: October 2004

Cell division in bacteria is mediated by the septal ring, a collection of about a dozen (known) proteins that localize to the division site, where they direct assembly of the division septum. The foundation of the septal ring is a polymer of the tubulin‐like protein FtsZ. Recently, experiments using fluorescence recovery after photobleaching have revealed that the Z ring is extremely dynamic. FtsZ subunits exchange in and out of the ring on a time scale of seconds even while the overall morphology of the ring appears static. These findings, together with in vitro studies of purified FtsZ, suggest that the rate‐limiting step in turnover of FtsZ polymers is GTP hydrolysis. Another component of the septal ring, FtsK, is involved in coordinating chromosome segregation with cell division. Recent studies have revealed that FtsK is a DNA translocase that facilitates decatenation of sister chromosomes by TopIV and resolution of chromosome dimers by the XerCD recombinase. Finally, two murein hydrolases, AmiC and EnvC, have been shown to localize to the septal ring of Escherichia coli, where they play an important role in separation of daughter cells.
Why is transcription coupled to translation in bacteria?
Authors: Gowrishankar J. , Harinarayanan R.
Published: October 2004

Active mechanisms exist to prevent transcription that is uncoupled from translation in the protein‐coding genes of bacteria, as exemplified by the phenomenon of nonsense polarity. Bacterial transcription–translation coupling may be viewed as one among several co‐transcriptional processes, including those for mRNA processing and export in the eukaryotes, that operate in the various life forms to render the nascent transcript unavailable for formation of otherwise deleterious R‐loops in the genome.
SsaM and SpiC interact and regulate secretion of Salmonella Pathogenicity Island 2 type III secretion system effectors and translocators
Authors: Yu Xiu‐Jun , Liu Mei , Holden David W.
Published: October 2004

The type III secretion system (TTSS) encoded by Salmonella Pathogenicity Island 2 (SPI‐2) is required for systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. The SPI‐2 TTSS is activated after internalization of bacteria by host cells, and translocates effector proteins into and across the vacuolar membrane, where they interfere with several host cell functions. Here, we investigated the function of SsaM, a small protein encoded within SPI‐2. An ssaM deletion mutant had virulence and intracellular replication defects comparable to those of a SPI‐2 TTSS null mutant. Although the ssaM mutant was able to secrete the effector protein SseJ in vitro, it failed to translocate SseJ into host cells, and to secrete the translocon proteins SseB, SseC and SseD in vitro. This phenotype is similar to that of a strain carrying a mutation in the SPI‐2 gene spiC, whose product is reported to be an effector involved in trafficking of the Salmonella vacuole in macrophages. Both ssaM and spiC mutants were found to oversecrete the SPI‐2 effector proteins SseJ and PipB in vitro. Fractionation assays and immunofluorescence microscopy were used to investigate the localization of SsaM and SpiC in macrophages. No evidence for translocation of these proteins was obtained. The similar phenotypes of the ssaM and spiC mutants suggested that they might be involved in the same function. Pull‐down and co‐immune precipitation experiments showed that SpiC and SsaM interact within the bacterial cell. We propose that a complex involving SsaM and SpiC distinguishes between translocators and effector proteins, and controls their ordered secretion through the SPI‐2 TTSS.
Regulation of FlbD activity by flagellum assembly is accomplished through direct interaction with the trans‐acting factor, FliX
Authors: Muir Rachel E. , Gober James W.
Published: October 2004

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the σ54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the trans‐acting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II‐encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1, does not alter the temporal pattern of FlbD‐dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two‐hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of σ54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD‐activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar‐flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.
Modulation of the mtrCDE‐encoded efflux pump gene complex of Neisseria meningitidis due to a Correia element insertion sequence
Authors: Rouquette‐Loughlin Corinne E. , Balthazar Jacqueline T. , Hill Stuart A. , Shafer William M.
Published: October 2004

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy‐dependent efflux pump system that is responsible for export of anti‐bacterial hydrophobic agents. Expression of the mtrCDE operon in gonococci is negatively regulated by the MtrR protein. Hydrophobic agent resistance mediated by the mtr system is also inducible, which results from an AraC‐like protein termed MtrA. In this work, we identified and characterized a pump similar to the gonococcal mtr system in various strains of Neisseria meningitidis. Unlike the situation with gonococci, the mtr system in meningococci is not subject to the MtrR or MtrA regulatory schemes. An analysis of the promoter region of the mtrCDE operon in a panel of meningococcal strains revealed the presence of one or two classes of insertion sequence elements. A 155–159 bp insertion sequence element known as the Correia element, previously identified elsewhere in the gonococcal and meningococcal genomes, was present in the mtrCDE promoter region of all meningococcal strains tested. In addition to the Correia element, a minority of strains had a tandemly linked, intact copy of IS1301. As described previously, a binding site for the integration host factor (IHF) was present at the centre of the Correia element upstream of mtrCDE genes. IHF was found to bind specifically to this site and deletion of the IHF binding site enhanced mtrC transcription. We also identified a post‐transcriptional regulation of the mtrCDE transcript by cleavage in the inverted repeat of the Correia element, as previously described by Mazzone et al. [Gene278: 211–222 (2001)] and De Gregorio et al. [Biochim Biophys Acta 1576: 39–44 (2002)]for other Correia element. We conclude that the mtr efflux system in meningococci is subject to transcriptional regulation by IHF and post‐transcriptional regulation by cleavage in the inverted repeat of the Correia element.
A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16‐3 is functional and convertible to suppressor tRNA
Authors: Blaha Béla , Semsey Szabolcs , Ferenczi Szilamér , Csiszovszki Zsolt , Papp Péter P. , Orosz László
Published: October 2004

Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16‐3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss‐of‐function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16‐3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16‐3, the proline tRNA gene retained its structural and functional integrity. Aspects of the co‐evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study.
The role of H‐NS in silencing F transfer gene expression during entry into stationary phase
Authors: Will William R. , Lu Jun , Frost Laura S.
Published: October 2004

The conjugative ability of the F plasmid of Escherichia coli is highly growth phase dependent, with plasmid transfer efficiency dropping rapidly as donor cells progress through the growth cycle towards stationary phase. Transfer is dependent on the expression of the plasmid transfer (tra) genes, which are controlled by three plasmid‐encoded regulatory proteins: TraJ, TraY and TraM. Here, we show that the nucleoid‐associated host protein, H‐NS, acts to repress the expression of traM and traJ as cells enter stationary phase, thereby decreasing mating ability to barely detectable levels. Sequence analysis identified regions of predicted intrinsic curvature, to which H‐NS preferentially binds, at the promoters of both traM and traJ. H‐NS binding at these regions was then confirmed by electrophoretic mobility shift and DNase I protection footprinting assays. Immunoblot assays displayed a significant increase in TraJ and TraM levels in an hns mutant strain. These findings were further supported by Northern and primer extension analyses which showed that whereas both genes were only expressed in early exponential phase in wild‐type cells, hns mutant cells exhibited drastic derepression throughout the growth cycle. Transcriptional fusion studies of the individual promoters demonstrated that H‐NS‐mediated repression was observed when the promoters of both traM and traJ were present in cis to each other. This suggests that H‐NS may bind to an extended region of the F plasmid, acting as a regional silencer of promoters for traJ and traM.
Characterization of an ABCA‐like transporter involved in vesicular trafficking in the protozoan parasite Trypanosoma cruzi
Authors: Torres Cristina , Pérez‐Victoria F. Javier , Parodi‐Talice Adriana , Castanys Santiago , Gamarro Francisco
Published: October 2004

Protozoan parasites are responsible of important healthy problems, among others malaria, leishmaniasis and trypanosomiasis. The present work reports the characterization of the first mammalian ATP‐binding cassette transporter, subfamily A (ABCA)‐like in Trypanosoma cruzi. TcABC1 is a single copy gene differentially expressed along the life cycle of the parasite, being absent in its infective form. TcABC1 localizes to the plasma membrane, flagellar pocket and intracellular vesicles. Functional studies of TcABC1 in transfected parasites suggest that the protein is implicated in intracellular trafficking, as determined by the analysis of endocytosis and exocytosis events. The accumulation of the endocytic markers FM4‐64 and NBD‐SM is increased in transfected parasites. Similarly, ectophosphatase and ectoATPase activities are increased in TcABC1 overproducers. Indeed, transmission electronic microscopy analysis showed a higher number of intracellular vesicles in TcABC1 transfectants. Taken together, these results suggest that the protein is involved in the endocytic and exocytic pathways of T. cruzi.
Structure and assembly of the pseudopilin PulG
Authors: Köhler Rolf , Schäfer Karsten , Müller Shirley , Vignon Guillaume , Diederichs Kay , Philippsen Ansgar , Ringler Philippe , Pugsley Anthony P. , Engel Andreas , Welte Wolfram
Published: October 2004

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram‐negative bacterium Klebsiella oxytoca. The sequence of the N‐terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X‐ray crystallography, was found to include part of the long N‐terminal α‐helix and the four internal anti‐parallel β‐strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left‐handed helical pili with the long N‐terminal α‐helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N‐terminal part of the PulG α‐helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.
Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein
Authors: Dean Paul , Kenny Brendan
Published: October 2004

The human intestinal pathogen, enteropathogenic Escherichia coli (EPEC), causes diarrhoeal disease by a mechanism that is dependent on the injection of effector proteins into the host cell. One effector, EspF, is reported to be required for EPEC to disrupt tight junction integrity of intestinal cells and increase the paracellular movement of molecules, which is likely to  contribute  to  diarrhoea.  Here,  we  show  that  not one but three EPEC‐encoded factors play important roles in this process. Thus, the Map (Mitochondria‐associated protein) effector is shown to: (i) be as essential as EspF for disrupting intestinal barrier function, (ii) be able to function independently of EspF, (iii) alter tight junction structure and (iv) mediate these effects in the absence of mitochondrial targeting. Additionally, the outer membrane protein Intimin is shown to be crucial for EspF and Map to disrupt the intestinal barrier function. This function of Intimin is completely independent of its interaction with its known receptor Tir, revealing a physiologically relevant requirement for Intimin interaction with alternative receptor(s). This work demonstrates that EPEC uses multiple multifunctional proteins to elicit specific responses in intestinal cells and that EPEC can control the activity of its injected effector molecules from its extracellular location.
Integration host factor is involved in transcriptional regulation of theBrucella abortus virBoperon
Authors: Sieira Rodrigo , Comerci Diego J. , Pietrasanta Lía I. , Ugalde Rodolfo A.
Published: October 2004

Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. ThevirBoperon ofBrucella abortuscodes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out usinglacZtranscriptional fusions showed thatvirBpromoter (PvirB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of thevirBoperon. Structural analyses showed that PvirBand regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind PvirBwas isolated and identified.  This  protein,  homologue  to  integration host factor (IHF), specifically interacts with PvirBand induces a DNA bending with an angle of 50.36°. DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with PvirB‐lacZfusions showed that inB. abortusIHF participates in the regulation of PvirBactivity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on thevirBoperon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellularvirBoperon expression being required for the biogenesis of the endoplasmic reticulum‐derived replicative vacuole.
Origin pairing (‘handcuffing’) and unpairing in the control of P1 plasmid replication
Authors: Das Nilangshu , Chattoraj Dhruba K.
Published: October 2004

The P1 plasmid origin has an array of five binding sites (iterons) for the plasmid‐encoded initiator protein RepA. Saturation of these sites is required for initiation. Iterons can also pair via their bound RepAs. The reaction, called handcuffing, is believed to be the key to control initiation negatively. Here we have determined some of the mechanistic details of the reaction. We show that handcuffed RepA–iteron complexes dissociate when they are diluted or challenged with cold competitor iterons, suggesting spontaneous reversibility of the handcuffing reaction. The complex formation increases with increased RepA binding, but decreases upon saturation of binding. Complex formation also decreases in the presence of molecular chaperones (DnaK and DnaJ) that convert RepA dimers to monomers. This indicates that dimers participate in handcuffing, and that chaperones are involved in reversing handcuffing. They could play a direct role by reducing dimers and an indirect role by increasing monomers that would compete out the weaker binding dimers from the origin. We propose that an increased monomer to dimer ratio is the key to reverse handcuffing.
Molecular approaches to malaria
Authors: Lingelbach Klaus , Kirk Kiaran , Rogerson Stephen , Langhorne Jean , Carucci Daniel J. , Waters Andrew
Published: October 2004

Malaria is a serious health problem in developing countries. With the complete sequencing of the genomes of the parasite and of the mosquito vector, malaria research has entered the post‐genome era. In this report we summarize the results and new research avenues presented at a recent meeting held with the aim of developing interdisciplinary approaches to combat this disease.
Two major histocompatibility complex haplotypes influence susceptibility to sporadic inclusion body myositis: critical evaluation of an association with HLA‐DR3
Authors: Price P. , Santoso L. , Mastaglia F. , Garlepp M. , Kok C.C. , Allcock R. , Laing N.
Published: October 2004

Abstract:  Previous studies of sporadic inclusion body myositis (sIBM) have shown a strong association with HLA‐DR3 and other components of the 8.1 ancestral haplotype (AH) (HLA‐A1, B8, DR3), where the susceptibility locus has been mapped to the central major histocompatibility complex (MHC) region between HLA‐DR and C4. Here, the association with HLA‐DR3 and other genes in the central MHC and class II region was further investigated in a group of 42 sIBM patients and in an ethnically similar control group (n = 214), using single‐nucleotide polymorphisms and microsatellite screening. HLA‐DR3 (marking DRB1*0301 in Caucasians) was associated with sIBM (Fisher's test). However, among HLA‐DR3‐positive patients and controls, carriage of HLA‐DR3 without microsatellite and single‐nucleotide polymorphism alleles of the 8.1AH (HLA‐A1, B8, DRB3*0101, DRB1*0301, DQB1*0201) was marginally less common in patients. Patients showed no increase in carriage of the 18.2AH (HLA‐A30, B18, DRB3*0202, DRB1*0301, DQB1*0201) or HLA‐DR3 without the central MHC of the 8.1AH, further arguing against HLA‐DRB1 as the direct cause of susceptibility. Genes between HLA‐DRB1 and HOX12 require further investigation. BTL‐II lies in this region and is expressed in muscle. Carriage of allele 2 (exon 6) was more common in patients. BTL‐II(E6)*2 is characteristic of the 35.2AH (HLA‐A3, B35, DRB1*01) in Caucasians and HLA‐DR1, BTL‐II(E6)*2, HOX12*2, RAGE*2 was carried by several patients. The 8.1AH and 35.2AH may confer susceptibility to sIBM independently or share a critical allele.
Genetic analysis of the CD28/CTLA4/ICOS (CELIAC3) region in coeliac disease
Authors: Amundsen S.S. , Naluai Å.T. , Ascher H. , Ek J. , Gudjónsdóttir A.H. , Wahlström J. , Lie B.A. , Sollid L.M.
Published: October 2004

Abstract:  In order to extend our previous findings of genetic linkage to the CD28/CTLA4/ICOS region on chromosome 2q33 (CELIAC3) in coeliac disease (CD), we have investigated 22 genetic markers in 325 Norwegian/Swedish multiplex and simplex CD families. We found both linkage and association with several markers, primarily in the multiplex material. We observed strong linkage disequilibrium (LD) between SNPs (Single Nucleotide Polymorphisms) within an LD block delimited by MH30 and D2S72. A haplotype of this region marked by the alleles −1147*T: + 49*A:CT60*G:CT61*A was significantly associated with CD, suggesting that one or more polymorphisms of this haplotype, possibly −1147*T, are involved in CD susceptibility. The CT60 SNP, a polymorphism found to be most strongly associated with some other immune‐mediated diseases, was not associated with CD, as this SNP was part of both associated and non‐associated haplotypes. Moreover, our results suggest that CELIAC3 harbours several independent loci contributing to CD susceptibility.
Integration of microsatellite characteristics in the MHC region: a literature and sequence based analysis
Authors: Gourraud P.A. , Mano S. , Barnetche T. , Carrington M. , Inoko H. , Cambon‐Thomsen A.
Published: October 2004

Abstract:  Reviews of microsatellite markers in the human leukocyte antigen region have been very useful in addressing the needs of the immunogenetics community. Nevertheless, characterization of the same microsatellite loci in different laboratories can lead to seemingly contradictory results, particularly in terms of nomenclature. Here we provide an update of previous reports, as well as a standardized characterization of primers for microsatellites located within the major histocompatibility complex (MHC). A uniform and extended inventory of 378 primer pairs from published reports was performed as well as a standardized characterization of the corresponding microsatellite loci according to the extended full‐length consensus sequence of MHC region. The literature‐based approach was complemented by a sequence‐based analysis of each reported microsatellite locus. Iterative electronic polymerase chain reaction runs and an original algorithm that characterizes patterns of repeats within sequence were used. The sequence of primers was corrected according to the consensus sequence. Table of synonymous names for individual microsatellite loci is provided.
Tetraspanin microdomains in immune cell signalling and malignant disease
Authors: Wright M.D. , Moseley G.W. , Van Spriel A.B.
Published: October 2004

Abstract:  A contemporary goal of researchers in leucocyte signalling has been to uncover how cells physically organize and compartmentalize signalling molecules into efficient, regulated signalling networks. This work has revealed important roles of membrane microdomains that are characterized by their distinctive protein and lipid compositions. Recent studies have demonstrated that besides typical cholesterol‐ and glycosphingolipid‐enriched ‘rafts’, leucocyte membranes are equipped with a different type of microdomain, made up of tetraspanin proteins. Tetraspanin proteins are involved in the organization of tetraspanin‐enriched microdomains by virtue of their capacity to specifically associate with key molecules, including integrins, leucocyte receptors and signalling proteins. The aspects of leucocyte function influenced by tetraspanin microdomains include adhesion, proliferation and antigen presentation. However, the mechanisms by which tetraspanin complexes link to intracellular signalling pathways, are still largely unknown. This review discusses how tetraspanin microdomains might function to regulate signalling in lymphoid and myeloid cells, and how they relate to lipid rafts. In addition, we discuss new insights into the role of tetraspanins in malignant disease.
New indolicidin analogues with potent antibacterial activity*
Authors: Ryge T.S. , Doisy X. , Ifrah D. , Olsen J.E. , Hansen P.R.
Published: October 2004

Abstract:  Indolicidin is a 13‐residue antimicrobial peptide amide, ILPWKWPWWPWRR‐NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two‐fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR‐NH2 in which Trp‐residues 4,6,8,9,11 were replaced in all positions with X = a single non‐natural building block; N‐substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non‐natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N‐terminus with a hydrophobic non‐natural building block. We prepared 22 analogues of indolicidin and [Phe4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non‐natural building block attached to the N‐terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non‐natural building blocks, are promising lead structures for developing future therapeutics.
Incorporation of vinylogous scaffolds in the C‐terminal tripeptide of substance P
Authors: Claudel S. , Tasseau O. , Sagan S. , Grison C. , Coutrot P. , Lavielle S.
Published: October 2004

Abstract:  Glycine‐9 and leucine‐10 of substance P (SP) are critical for (NK)‐1 receptor recognition and agonist activity. Proψ(Z)‐CH=CH(CH3)‐CONH)Leu (or Met) and Proψ((E)‐CH=CH(CH3)‐CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C‐terminal tripeptide, Gly‐Leu‐Met‐NH2, of SP. Proψ((Z)‐CH=C(CH2CH(CH3)2)‐CONH)Met‐NH2, with an isobutyl substituent to mimic the Leu side‐chain, was also incorporated in place of the C‐terminal tripeptide. The substituted‐SP analogs were tested for their affinity to human NK‐1 receptor specific binding sites (NK‐1M and NK‐1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK‐1 receptor. The most potent SP analogs [Pro9ψ((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9ψ ((E)CH=C(CH3)CONH)Leu10]SP, are about 100‐fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)‐ or (E)‐CH=C(CH3)‐ or (Z)‐CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C‐terminal tripeptide of SP within both the NK‐1M‐ and NK‐1m‐specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.
A napin‐like polypeptide with translation‐inhibitory, trypsin‐inhibitory, antiproliferative and antibacterial activities from kale seeds
Authors: Ngai P.H.K. , Ng T.B.
Published: October 2004

Abstract:  A heterodimeric napin‐like polypeptide with translation‐inhibiting and antibacterial activities has been isolated from kale seeds. The purification procedure entailed ion‐exchange chromatography on dielthylaminoethyl (DEAE)‐cellulose, affinity chromatography on Affi‐gel blue gel, ion‐exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. The napin‐like polypeptide was unadsorbed on DEAE‐cellulose but adsorbed on Affi‐gel blue gel and Mono S. Its 7‐kDa large subunit differs in N‐terminal amino acid sequence from the 4‐kDa small subunit. The polypeptide inhibited translation in the rabbit reticulocyte lysate system with an IC50 of 37.5 nm. This activity was preserved between pH 5 and pH 11, and between 10 and 40 °C. It fell to a low level at pH 3 and pH 13 and at 70 °C. Antibacterial activity against Bacillus, Megabacterium, and Pseudomonas species and antiproliferative activity against leukemia L1210 cells were observed. However, the polypeptide did not exert antifungal, ribonuclease, or protease activity.
Transmission of Streptococcus mutans in a group of Turkish families
Authors: Ersin N. K. , Kocabas E. H. , Alpoz A. R. , Uzel A.
Published: October 2004

Background/aims:  To investigate the transmission of Streptococcus mutans in a group of Turkish families using AP‐polymerase chain reaction (PCR) detection.

Methods:  Eight mothers who had high S. mutans levels in unstimulated saliva and 8 children aged between 2 and 3 years participated in the study. Plaque samples from each child were collected with the tips of sterile toothpicks for S. mutans counts. Although not part of the original study design, S. mutans samples were also obtained from the unstimulated saliva of the three fathers who shared the same households. Three typical isolates of S. mutans were isolated from TYCSB agar of each subject and identified by sugar fermentation tests. S. mutans ATCC 10449 was used as the reference strain. AP‐PCR was conducted with OPA‐05 primer.

Results:  All of the mothers and fathers shared the similar genotypes within their children. The fathers also harbored similar genotypes to their spouses.

Conclusion:  The mothers or the fathers could be the source for the transmission of S. mutans to their children.
Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by denaturing gradient gel electrophoresis fingerprinting approach
Authors: Siqueira J. F. , Rôças I. N. , Rosado A. S.
Published: October 2004

The purpose of the present study was to investigate the bacterial communities associated with asymptomatic and symptomatic endodontic infections and to compare denaturing gradient gel electrophoresis (DGGE) fingerprinting patterns of these two clinical conditions. The root canal microbiota of teeth associated with asymptomatic or symptomatic periradicular lesions was profiled by the PCR‐DGGE method and then compared, taking into consideration the banding patterns. Bacteria were present in all examined cases. Comparative analysis of the two clinical conditions revealed bands that were common to both symptomatic and asymptomatic cases, but most DGGE bands appeared to be unique for each clinical condition. No single band occurred in all profiles. The mean number of bands detected in the 16S rDNA community profiles were 12.1 ± 9.4 (range 2–29) for symptomatic samples and 6.7 ± 2.7 (range 2–11) for asymptomatic ones. Clustering methods and principal component analysis of DGGE banding pattern placed the samples according to the presence or absence of symptoms. Four intense bands that were excised from the gel and sequenced showed similarities to species of the Campylobacter genus (found in 5/12 asymptomatic and in 3/11 symptomatic cases), Fusobacterium genus (4/11 symptomatic cases), Acinetobacter genus (5/12 asymptomatic cases), and Enterobacteriaceae family (11/12 asymptomatic and 2/11 symptomatic cases). The profiles of the predominant bacterial community appeared to be unique for each individual. These findings confirm that endodontic infections are polymicrobial and showed that there are significant differences in the predominant bacterial composition between asymptomatic and symptomatic cases.
Detection frequency of periodontitis‐associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects
Authors: Mayanagi G. , Sato T. , Shimauchi H. , Takahashi N.
Published: October 2004

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (≥50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002≤P<0.05). Two species (Mogibacterium timidum and Porphyromonas gingivalis) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects (P<0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.
Role of a Streptococcus gordonii copper‐transport operon, copYAZ, in biofilm detachment
Authors: Mitrakul K. , Loo C. Y. , Hughes C. V. , Ganeshkumar N.
Published: October 2004

Streptococcus gordonii is a pioneer oral bacterium that is associated with the initiation of dental plaque development. Located downstream of the S. gordonii adc operon, which is involved in competence and biofilm formation, were three open reading frames, designated copY, copA and copZ. These open reading frames were homologous to the copYAZ genes in Streptococcus mutans that are involved in copper homeostasis and biofilm detachment. This study examined whether copYAZ genes play any role in the biofilm formation and detachment of S. gordonii. The copY gene encodes a 143‐amino acid protein homologous to the negative transcriptional regulator of a copper‐transport operon, copA encodes a 748‐amino acid copper‐transporting P‐type ATPase, and copZ encodes a 69‐amino acid putative metallochaperone protein in S. mutans. Each open reading frame in the copYAZ operon in S. gordonii was inactivated by insertional mutation and the growth, biofilm formation and detachment of each mutant were examined. S. gordonii copY::specR, copA::specR, and copZ::specR mutants were able to form biofilms on both polystyrene and glass surfaces. However, inactivation of copZ and to a lesser extent copY resulted in phenotypes that were defective in biofilm detachment, which is consistent with previous observations in S. mutans and suggests that the trace element copper might influence biofilm detachment of bacterial biofilms.
Effect of Fruiting Traits on the Field Resistance of Cocoa (Theobroma cacao L.) Clones to Phytophthora megakarya
Authors: Efombagn M. I. B. , Marelli J. P. , Ducamp M. , Cilas C. , Nyassé S. , Vefonge D.
Published: October 2004

The effects of some traits of field resistance (precocity and duration of the fruiting cycle, age of diseased fruit and vertical pod distribution on the tree) to Phytophthora megakarya of four known cocoa clones were studied in an on‐station clonal plot planted in 1982 in the south‐west of Cameroon. Weekly observations of fruit set and development, black pod and rainfall were carried out during three growing seasons (1999, 2000 and 2001). The study confirmed the previous field and laboratory assessments of resistance of these clones based on the mean percentages of rotten pods obtained annually. The present study has permitted the identification of fruit aged 2–3 months as the highly susceptible stage of development in the most susceptible clone. In addition, precocity and pod cycle duration varied significantly among the clones. The earlier the pod cycle began, the more susceptible was the clone: the most resistant clone started flowering 1 month after the most susceptible clone and therefore escaped the peak of disease severity. Rainfall intensity greatly modified the incidence of the disease in 2001, with high yield losses occurring in all four clones (70–93%), but their ranking remained stable over the 3 years. The spatial distribution of pods on the trees showed that pods on the trunk were more likely to become diseased than those on the branches, but its effect as a clone resistance component is variable among the four clones; the resistant clone producing more pods on the trunk and the susceptible clone more in the canopy.
Virulence–Toxin Production Relationship in Isolates of the Plant Pathogenic Fungus Botrytis cinerea
Authors: Reino J. L. , Hernández‐Galán R. , Durán‐Patrón R. , Collado I. G.
Published: October 2004

Eleven isolates of Botrytis cinerea were studied to examine the relationship between toxin production and virulence. After 5 days of incubation, screening experiments revealed significant differences in toxin production by the strains. The isolates with low toxin production were less virulent; moreover, the only toxins isolated were those corresponding to botrydial or its derivatives. In contrast, higher amounts of toxins were isolated from the more aggressive isolates. Furthermore, two classes of toxins, those with botryane skeleton and botcinolide derivatives, were detected in and isolated from all aggressive strains studied. This indicates that a synergistic action of several toxins is involved in the phytotoxicity of this phytopathogen.
Agronomic and chemical characterization of 39 Hypericum perforatum accessions between 1998 and 2000
Authors: Poutaraud A. , Girardin P.
Published: October 2004

Thirty‐nine accessions of St John's Wort were studied over a 3‐year period. The percentage of diseased plants ranged from 0 to 100%. After 3 years of cultivation, 18% of the accessions presented a dieback rate of <10%. Hypericins and hyperforins in flowering tops (top 30 cm of plants at full bloom) and flowers were assayed using high‐performance liquid chromatography (HPLC‐DAD Diode Array Detector) (one to three cuttings a year). The hypericin contents in flowering tops ranged from 0.7 to 3%. These levels were, on average, 2.8‐fold lower than those recorded in flowers. Hyperforin levels varied in the different accessions, ranging from 0.65 to 3% in flowering tops and 2 to 5.7% in the flowers. In line with present industrial needs, a minimum content of one or more of the active components, it will therefore be necessary to select accessions in terms of the dry weight yield and the contents of flowering tops and flowers. Dry weight depends on cutting height, which also affects the quality of the plant material harvested. Because of the plant to plant variability, the performance of the accessions selected would be improved.