bluuebunny/crossref_metadata_2025 huggingface dataset:
Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples
Authors: McHugh T D , Pope C F , Ling C L , Patel S , Billington O J , Gosling R D , Lipman M C , Gillespie S H
Published: November 2004

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis.
Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing
Authors: Lau Susanna KP , Woo Patrick CY , Fung Ami MY , Chan King-man , Woo Gibson KS , Yuen Kwok-yung
Published: November 2004

Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood. The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described. The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported. Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing. Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O. uli. Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta. Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P. acnes and polymicrobial bacteraemia were associated with pseudobacteraemia. Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common. The overall mortality rate was 31 %. Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P. acnes should be treated with appropriate antibiotics. The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of high pathogenicity. Further studies to look for specific virulence factors are warranted.
Changes in renal haemodynamics induced by indomethacin in the rat involve cytochrome P450 arachidonic acid‐dependent epoxygenases‡
Authors: Caron N , El Hajjam A , Declèves A‐E , Joly E , Falck JR , Kramp R
Published: November 2004

1. A significant renal vasodilation was observed previously after an acute cyclo‐oxygenase (COX) inhibition induced with indomethacin. Because this effect could be due to COX‐dependent intrarenal metabolization of arachidonic acid through cytochrome P450 (CYP450) pathways, the aim of the present study was to investigate, in vivo, possible interactions between COX and CYP450 mono‐oxygenases.

2. Mean arterial pressure (MAP) and renal blood flow (RBF), using an electromagnetic flow transducer for RBF evaluation, were measured continuously in 71 anaesthetized euvolaemic rats. Appropriate solvents (vehicle), 3 mg/kg indomethacin, 17‐octadecynoic acid (17‐ODYA; 2 mmol/L), either miconazole (MI; 1.5 mmol/L) or N‐methylsulphonyl‐6‐(2‐propargyloxyphenyl)hexanamide (MS‐PPOH; 5 mg/kg) and N′‐hydroxyphenylformamidine (HET0016; 5 or 10 mg/kg) were administered to inhibit either COX, CYP450 mono‐oxygenases, epoxygenases or hydroxylase, respectively. The CYP450 and COX inhibitors were also combined as follows: 17‐ODYA/indomethacin, MI (or MS‐PPOH)/indomethacin, HET0016/indomethacin and indomethacin/HET0016.

3. Mean arterial pressure and RBF were not modified by vehicle, 17‐ODYA or MI (or MS‐PPOH). However, MAP decreased slightly (P < 0.05; paired t‐test, 5 d.f.) and RBF increased transiently (P < 0.05; anova, 5 d.f.) after HET0016. In contrast, MAP decreased by 10 mmHg (P < 0.05) and RBF increased by 10% (P < 0.05) after indomethacin. This enhancement was prevented by 17‐ODYA or MI (or MS‐PPOH), but not by HET0016. Moreover, RBF increased step‐wise to 21% in the indomethacin/HET0016 experiment (P < 0.05). Consequently, changes from baseline in renal vascular resistance differed among treatments, averaging −2 ± 3 (vehicle), −13 ± 3 (indomethacin; P < 0.05 vs vehicle), −4 ± 3 (17‐ODYA/indomethacin), −3 ± 4 (MI or MS‐PPOH/indomethacin), −15 ± 3 (HET0016/indomethacin; P < 0.05) and −22 ± 4% (indomethacin/HET0016; P < 0.05).

4. In conclusion, these results demonstrate that the renal vasodilation induced by indomethacin can be prevented by prior inhibition of CYP450 mono‐oxygenases and further suggest that the CYP450 epoxygenases pathway may prevail.
‘The Bombay Debt’: Letter Writing, Domestic Economies and Family Conflict in Colonial India
Authors: Rappaport Erika
Published: November 2004

Between 1856 and 1861 Minnie Blane and her husband, Captain Archibald Wood, wrote dozens of letters from India to the Minnie's mother in England. These letters and those associated with a military investigation into the couple's relationship in the 1860s detail the connections between the breakdown of the East India Company's rule in India and Minnie Blane's marriage. In particular, this correspondence shows some of the ways in which bourgeois identities were constructed in relationship to money and objects, place and race. It also exposes the fissures between family members, allowing us to see the gender, generational and cultural conflicts within such imperial families. The article raises concerns about the ways in which personal letters have been used as documents in the study of European women's imperial history.
The Autocracy of Love and the Legitimacy of Empire: Intimacy, Power and Scandal in Nineteenth‐Century Metlakahtlah
Authors: Perry Adele
Published: November 2004

This paper examines the politics of intimacy, power, and scandal at Metlakahtlah, a Church of England mission village in northern British Columbia, Canada, from 1862 to 1885, in order to cast light on settler colonialism and its aftermath. It particularly examines Metlakahtlah's main missionary, William Duncan, his relationships with young female converts and missionary women, and, perhaps more importantly, the stories that were told about them. Stories of Duncan's relationships with young Tsimshian women that circulated throughout settler society reveal the central place of sexuality to both critiques and defences of imperialism, and cast new light on contemporary politics around the historical experience of Indigenous children in settler colonies like Australia and Canada.
Celebrating the Ordinary: Church, Empire and Gender in the Life of Mère Marie‐Michelle Dédié(Senegal, Congo, 1882–1931)
Authors: Martin Phyllis M.
Published: November 2004

Member of the congregation of the Sisters of Saint Joseph of Cluny, Mère Marie‐Michelle Dédié worked within a highly gendered institution, the Catholic church and within a highly gendered colonial society. Although at considerable personal cost, she successfully achieved her goals – the training of African girls and women in the Christian life – by working within prescribed boundaries rather than contesting them. She was lauded by churchmen for her dedication and fortitude, and celebrated by colonial men, who found and imagined in her the kinder face of the imperial enterprise. Mère Marie's life suggests a study of the lives of missionary nuns in the outposts of empire may add to our understanding of the contradictions of empire and the complexities of colonial society, as well as the predicaments and successes of religious women.
Looking for Feminism
Authors: Sarkar Mahua
Published: November 2004

This paper explores the linkages between metropolitan intellectual politics and the process of knowledge production within third‐world/anti‐imperial locations – defined geographically or otherwise – about previously understudied subjects from or in the third world. Specifically, the paper focuses on the growing body of research on Muslim women in colonial India to foreground ways in which a certain notion of feminism, as a metanarrative of emergence and progress towards an apparently known end, informs and shapes this literature. In terms of its broader implications, the paper is an attempt to think through some of the problems of writing histories of difference, and especially feminist histories, in any context.
The Wealth Effects of Sale and Leasebacks: New Evidence
Authors: Fisher Lynn M.
Published: November 2004

This paper investigates the phenomenon of sale and leasebacks as one way in which firms may use financial contracts to rearrange their organizational architecture. A theoretic model links the length of initial leaseback period to incentives to make noncontractible future investments in the lease relationship and predicts that firms choose shorter leases when landlords make relatively important investments. Using a sample of 71 sale and leaseback events from the 1990s, we document a significant mean abnormal return of 1.3% for shareholders of seller/lessee firms announcing relatively short leasebacks. The evidence suggests that firms may use sale and leasebacks to optimize their claims to real estate.
The Pricing of the Emergent Leasehold (Possessory) Estates of Ghana
Authors: Asabere Paul K.
Published: November 2004

The paper develops hedonic analyses of the pricing of leasehold versus freehold estates in Ghana. The motivation of the paper is the passage of Act 267(5) in 1992 that effectively abolished outright sale of stool lands in Ghana. Stool lands are lands controlled by tribal “chiefs.” Act 267(5) prohibits the sale of freehold estates for stool lands. There are two important findings in this study. The first is that freeholds tend to attract premium prices relative to leaseholds. This supports the bundle‐of‐rights argument. The second is that the effect of the 1992 Act is indeed capitalized into land prices because of the relative increase in the supply of leaseholds vis‐à‐vis freeholds. Specifically, the price of freeholds increased relative to the price of leaseholds after the constitutional event. The study also finds that transactions involving stools and individuals, usually perceived to be associated with litigation risk (or title insecurity) relative to government lands, are sold at a discount. Transactions noted to have a history of litigation are also associated with price discounts. The estimated coefficients on all the variables representing services to the site like water, electricity and access roads are significantly positive, indicating a high demand for such essential services.
Neutrophils secrete MIP‐1β after adhesion to laminin contained in basement membrane of blood vessels
Authors: Chiba Kouji , Zhao Wenli , Chen Jiang , Wang Jingxin , Cui Hong Yan , Kawakami Hiroshi , Miseki Tetsuya , Satoshi Hashino , Tanaka Junji , Asaka Masahiro , Kobayashi Masanobu
Published: November 2004

We have recently demonstrated that granulocyte colony‐stimulating factor (G‐CSF) stimulated the production of epithelial‐cell‐derived‐neutrophil attractant‐78 (ENA‐78) by neutrophils and that ENA‐78 might promote the accumulation of neutrophils that had migrated from the intravascular space into inflammatory tissues. In this study, we examined whether other chemokines could be secreted by neutrophils that had accumulated after migrating from the intravascular space into the inflammatory tissues. We demonstrated that adhesion to laminin contained in the basement membrane and Matrigel, which is an artificial basement membrane model, induced macrophage inflammatory protein‐1β (MIP‐1β) in neutrophils and that MIP‐1β secreted by neutrophils induced the chemotaxis of dendritic cells. These findings suggest that neutrophils transmigrating through the basement membrane are stimulated to secrete MIP‐1β by the basement membrane, inducing the transmigration of dendritic cells from the intravascular space into inflammatory tissues. We propose that neutrophils intervene between innate immune response and specific immune response by secreting MIP‐1β during the transmigration through the basement membrane.
Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization
Authors: Rodriguez Marie‐Hélène , Plantier Jean‐Luc , Enjolras Nathalie , Réa Muriel , Leboeuf Marylène , Uzan Georges , Négrier Claude
Published: November 2004

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue‐specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B‐domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte‐produced FVIII.
The contribution of classical risk factors to cardiovascular disease in familial hypercholesterolaemia: data in 2400 patients
Authors: Jansen A. C. M. , van Aalst‐Cohen E. S. , Tanck M. W. , Trip M. D. , Lansberg P. J. , Liem A. H. , Roeters van Lennep H. W. O. , Sijbrands E. J. G. , Kastelein J. J. P.
Published: November 2004

Objective.  To determine the contribution of classical risk factors to the development of cardiovascular disease (CVD) in patients with heterozygous familial hypercholesterolaemia (FH).

Design.  A retrospective, multi‐centre, cohort study. Extensive data were collected by scrutinizing medical records and the use of questionnaires. Multivariate Cox regression was used to study the relationship between potential risk factors and the occurrence of CVD.

Setting and subjects.  We included 2400 FH patients from 27 Dutch lipid clinics. The diagnosis of FH was based upon the presence of a low‐density lipoprotein receptor mutation or upon strict clinical criteria.

Main outcome measures.  Cardiovascular mortality and CVD.

Results.  During 112.943 person‐years, 782 (32.6%) patients had had at least one cardiovascular event. Male gender (RR 2.82, 95% CI 2.37–3.36), smoking (RR 1.67, 95% CI 1.40–1.99), hypertension (RR 1.36, 95% CI 1.06–1.75), diabetes mellitus (RR 2.19, 95% CI 1.36–3.54), low HDL‐C (RR 1.37, 95% CI 1.15–1.63) and elevated lipoprotein(a) levels (RR 1.50, 95% CI 1.20–1.79) proved to be independent CVD risk factors. These six risk factors explained 18.7% of the variation in the occurrence of CVD.

Conclusions.  Male gender, smoking, hypertension, diabetes mellitus, HDL cholesterol and lipoprotein(a) levels proved to be important risk factors for CVD in FH patients. In addition to the routine instiQJ;tution of statin therapy, controlling these factors needs special attention in the management of this disorder.
Protein–protein interactions within the Fatty Acid Synthase‐II system of Mycobacterium tuberculosis are essential for mycobacterial viability
Authors: Veyron‐Churlet Romain , Guerrini Olivier , Mourey Lionel , Daffé Mamadou , Zerbib Didier
Published: November 2004

Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug‐resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front‐line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase‐II (FAS‐II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti‐tuberculous agents. Several data, in the literature, suggest the existence of protein–protein interactions within the FAS‐II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two‐hybrid approach and in vitro co‐immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS‐II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS‐II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS‐II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non‐pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS‐II. They also represent an important step towards the design of a new generation of anti‐tuberculous agents, as being inhibitors of essential protein–protein interactions.
An alternative pathway for reduced folate biosynthesis in bacteria and halophilic archaea
Authors: Levin Itay , Giladi Moshe , Altman‐Price Neta , Ortenberg Ron , Mevarech Moshe
Published: November 2004

Whereas tetrahydrofolate is an essential cofactor in all bacteria, the gene that encodes the enzyme dihydrofolate reductase (DHFR) could not be identified in many of the bacteria whose genomes have been entirely sequenced. In this communication we show that the halophilic archaea Halobacterium salinarum and Haloarcula  marismortui  contain  genes  coding for proteins with an N‐terminal domain homologous to dihydrofolate synthase (FolC) and a C‐terminal domain homologous to dihydropteroate synthase (FolP). These genes are able to complement a Haloferax volcanii mutant that lacks DHFR. We also show that the Helicobacter pylori dihydropteroate synthase can complement an Escherichia coli mutant that lacks DHFR. Activity resides in an N‐terminal segment that is homologous to the polypeptide linker that connects the dihydrofolate synthase and dihydropteroate synthase domains in the haloarchaeal enzymes. The purified recombinant H. pylori dihydropteroate synthase was found to be a flavoprotein.
Mitochondrial β‐oxidation in Aspergillus nidulans
Authors: Maggio‐Hall Lori A. , Keller Nancy P.
Published: November 2004

β‐Oxidation (β‐ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial β‐ox. Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial β‐ox. While growth of a peroxisomal β‐ox disruption mutant (ΔfoxA) was eliminated on a very long‐chain fatty acid (C22:1), growth was only partially impeded on a long‐chain fatty acid (C18:1) and was not affected at all on short chain (C4–C6) fatty acids. In contrast, growth of a putative enoyl‐CoA hydratase mutant (ΔechA) was abolished on short‐chain and severely restricted on long‐ and very long‐chain fatty acids. Furthermore fatty acids inhibited growth of the ΔechA mutant but not the ΔfoxA mutant in the presence of an alternate carbon source (lactose). Disruption of echA led to a 28‐fold reduction in 2‐butenoyl‐CoA hydratase activity in a preparation of organelles. EchA was also required for growth on isoleucine and valine. The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.
Bacterial subcellular architecture: recent advances and future prospects
Authors: Lewis Peter J.
Published: November 2004

Traditional textbook representations of the prokaryotic cytoplasm show an amorphous, unstructured amalgamation of proteins and small molecules in which a randomly arranged chromosome resides. The development and application of a swathe of microscopic techniques over the last 10 years in particular, has shown this image of the microbial cell to be incorrect: the cytoplasm is highly structured with many proteins carrying out their assigned functions at specific subcellular locations; bacteria contain cytoskeletal elements including microtubule, actin and intermediate filament homologues; the chromosome is not randomly folded and is organized in such a way as to facilitate efficient segregation upon cell division. This review will concentrate on recent advances in our understanding of subcellular architecture and the techniques that have led to these discoveries.
The related effector proteins SopD and SopD2 from Salmonella enterica serovar Typhimurium contribute to virulence during systemic infection of mice
Authors: Jiang Xiuju , Rossanese Olivia W. , Brown Nat F. , Kujat‐Choy Sonya , Galán Jorge E. , Finlay B. Brett , Brumell John H.
Published: November 2004

Salmonella resides within host cells in a vacuole that it modifies through the action of virulence proteins called effectors. Here we examined the role of two related effectors, SopD and SopD2, in Salmonella pathogenesis. Salmonella enterica serovar Typhimurium (S. Typhimurium) mutants lacking either sopD or sopD2 were attenuated for replication in the spleens of infected mice when competed against wild‐type bacteria in mixed infection experiments. A double mutant lacking both effector genes did not display an additive attenuation of virulence in these experiments. The double mutant also competed equally with both of the single mutants. Deletion of either effector impaired bacterial replication in mouse macrophages but not human epithelial cells. Deletion of sopD2 impaired Salmonella’s ability to form tubular membrane filaments [Salmonella‐induced filaments (Sifs)] in infected cells; the number of Sifs decreased, whereas the number of pseudo‐Sifs (thought to be a precursor of Sifs) was increased. Transfection of HeLa cells with the effector SifA induced the formation of Sif‐like tubules and these were observed in greater size and number after co‐transfection of SifA with SopD2. In infected cells, SifA and SopD2 were localized both to Sifs and to pseudo‐Sifs. In contrast, deletion of sopD had no effect on Sif formation. Our results indicate that both SopD and SopD2 contribute to virulence in mice and suggest a functional relationship between these two proteins during systemic infection of the host.
Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion
Authors: Atmakuri Krishnamohan , Cascales Eric , Christie Peter J.
Published: November 2004

Bacteria use type IV secretion systems (T4SS) to translocate DNA (T‐DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T‐DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP‐dependent mechanism. By co‐immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co‐synthesis of previously identified ‘core’ components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.
Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin
Authors: Schlünzen Frank , Pyetan Erez , Fucini Paola , Yonath Ada , Harms Jörg M.
Published: November 2004

Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 Å structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A‐tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P‐tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.
Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p
Authors: Bensen Eric S. , Martin Samuel J. , Li Mingchun , Berman Judith , Davis Dana A.
Published: November 2004

The human pathogen Candida albicans grows and colonizes sites that can vary markedly in pH. The pH response in C. albicans is governed in part  by the Rim101p pathway. In Saccharomyces cerevisiae, Rim101p promotes alkaline responses by repressing expression of NRG1, itself a transcriptional repressor. Our studies reveal that in C. albicans, Rim101p‐mediated alkaline adaptation is not through repression of CaNRG1. Furthermore, our studies suggest that Rim101p and Nrg1p act in parallel pathways to regulate hyphal morphogenesis, an important contributor to virulence. To determine the wild‐type C. albicans transcriptional response to acidic and alkaline pH, we utilized microarrays and identified 514 pH‐responsive genes. Of these, several genes involved in iron acquisition were upregulated at pH 8, suggesting that alkaline pH induces iron starvation. Microarray analysis of rim101–/– cells indicated that Rim101p does not govern transcriptional responses at acidic pH, but does regulate a subset of transcriptional responses at alkaline pH, including the iron acquisition genes. We found that rim101–/– cells are sensitive to iron starvation, which suggests that one important aspect of the Rim101p‐dependent alkaline pH response is to adapt to iron starvation conditions.
A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi
Authors: Seshu J. , Boylan Julie A. , Hyde Jenny A. , Swingle Kristen L. , Gherardini Frank C. , Skare Jonathan T.
Published: November 2004

Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non‐infectious B. burgdorferi isolate exhibited hypersensitivity to t‐butyl hydroperoxide when compared with infectious B. burgdorferi and other non‐infectious isolates. Sequence analysis indicated that the hypersensitive non‐infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non‐infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t‐butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter–lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K  synthesized  fourfold  greater  levels  of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.
The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels
Authors: Datta Kaustuv , Skidmore Jennifer M. , Pu Kun , Maddock Janine R.
Published: November 2004

The Obg subfamily of bacterial GTP‐binding proteins are biochemically distinct from Ras‐like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras‐like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP‐binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature‐sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non‐permissive temperature, G80E  cells  rapidly  lose  viability  and  yet  do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1‐S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.
Genetic recombination and the cell cycle: what we have learned from chromosome dimers
Authors: Lesterlin Christian , Barre François‐Xavier , Cornet François
Published: November 2004

Genetic recombination is central to DNA metabolism. It promotes sequence diversity and maintains genome integrity in all organisms. However, it can have perverse effects and profoundly influence the cell cycle. In bacteria harbouring circular chromosomes, recombination frequently has an unwanted outcome, the formation of chromosome dimers. Dimers form by homologous recombination between sister chromosomes and are eventually resolved by the action of two site‐specific recombinases, XerC and XerD, at their target site, dif, located in the replication terminus of the chromosome. Studies of the Xer system and of the modalities of dimer formation and resolution have yielded important knowledge on how both homologous and site‐specific recombination are controlled and integrated in the cell cycle. Here, we briefly review these advances and highlight the important questions they raise.
The C‐terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities
Authors: Sztukowska Maryta , Sroka Aneta , Bugno Marcin , Banbula Agnieszka , Takahashi Yusuke , Pike Robert N. , Genco Caroline A. , Travis James , Potempa Jan
Published: November 2004

The Porphyromonas gingivalis lysine‐specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro‐fragment, a catalytic domain with a C‐terminal IgG‐like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non‐catalytic domains in pro‐Kgp processing and maturation in P. gingivalis, the wild‐type form of the gene was replaced with deletion variants encoding C‐terminally truncated proteins, including KgpΔHA3/4 (Δ1292–1732 aa), KgpΔHA2‐4 (Δ1157–1732 aa), KgpΔHA1‐4 (Δ738–1732 aa), KgpΔC‐term/HA (Δ681–1732 aa) and KgpΔIg/C‐term/HA (602–1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT‐PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpΔC‐term/HA and kgpΔIg/C‐term/HA transcripts, no Kgp‐specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp‐specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane‐associated and ‐secreted forms. While both gingipain K activity and the protein were cell membrane‐associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C‐terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin‐like subdomain is indispensable for proper folding and expression of the gingipains.
Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem–loop structure
Authors: Venkova‐Canova Tatiana , Soberón Nora E. , Ramírez‐Romero Miguel A. , Cevallos Miguel A.
Published: November 2004

The repABC replicons contain an operon encoding the initiator protein (RepC) and partitioning proteins (RepA and RepB). The latter two proteins negatively regulate the transcription of the operon. In this article we have identified two novel regulatory elements, located within the conserved repB–repC intergenic sequence, which negatively modulate the expression of repC, in plasmid p42d of Rhizobium etli. One of them is a small antisense RNA and the other is a stem–loop structure in the repABC mRNA that occludes the Shine‐Dalgarno sequence of repC. According to in vivo and in vitro analyses, the small antisense RNA (57–59 nt) resembles canonical negative regulators of replication because: (i) it is transcribed from a strong constitutive promoter (P2), (ii) the transcript overlaps untranslated region upstream of the RepC coding sequences, (iii) the RNA forms one secondary structure acting as a rho‐independent terminator, (iv) the antisense RNA is a strong trans‐incompatibility factor and (v) its presence reduces the level of repC expression. Surprisingly, both of these seemingly negative regulators are required for efficient plasmid replication.
The Campylobacter jejuni dccRS two‐component system is required for optimal in vivo colonization but is dispensable for in vitro growth
Authors: MacKichan Joanna K. , Gaynor Erin C. , Chang Christopher , Cawthraw Shaun , Newell Diane G. , Miller Jeff F. , Falkow Stanley
Published: November 2004

A Campylobacter jejuni two‐component signal transduction system (TCSTS), designated dccR‐dccS (diminished capacity to colonize; Cj1223c‐Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A ΔdccR response regulator mutant generated using the virulent strain 81–176 background exhibited significantly reduced colonization of immunocompetent limited flora (I‐LF) mice, severe combined immunodeficient limited flora (SCID‐LF) mice, and 1‐day‐old chicks. A ΔdccS sensor kinase mutant was likewise defective for colonization in the I‐LF mouse model. ΔdccR‐infected SCID‐LF mice also exhibited dramatically reduced inflammation relative to wild type‐infected SCID‐LF mice. Despite this diminished colonization capacity, the ΔdccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two‐component system; binding of purified His‐tagged DccR to the promoter region of two of these genes supports a direct protein–DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two‐component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.
Models and mechanisms of O‐O bond activation by cytochrome P450
Authors: Hlavica Peter
Published: November 2004

Cytochrome P450 enzymes promote a number of oxidative biotransformations including the hydroxylation of unactivated hydrocarbons. Whereas the long‐standing consensus view of the P450 mechanism implicates a high‐valent iron‐oxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound pathway, more recent studies on isotope partitioning, product rearrangements with ‘radical clocks’, and the impact of threonine mutagenesis in P450s on hydroxylation rates support the notion of the nucleophilic and/or electrophilic (hydro)peroxo‐iron intermediate(s) to be operative in P450 catalysis in addition to the electrophilic oxenoid‐iron entity; this may contribute to the remarkable versatility of P450s in substrate modification. Precedent to this mechanistic concept is given by studies with natural and synthetic P450 biomimics. While the concept of an alternative electrophilic oxidant necessitates C‐H hydroxylation to be brought about by a cationic insertion process, recent calculations employing density functional theory favour a ‘two‐state reactivity’ scenario, implicating the usual ferryl‐dependent oxygen rebound pathway to proceed via two spin states (doublet and quartet); state crossing is thought to be associated with either an insertion or a radicalar mechanism. Hence, challenge to future strategies should be to fold the disparate and sometimes contradictory data into a harmonized overall picture.
Molecular evolution of shark and other vertebrate DNases I
Authors: Yasuda Toshihiro , Iida Reiko , Ueki Misuzu , Kominato Yoshihiko , Nakajima Tamiko , Takeshita Haruo , Kobayashi Takanori , Kishi Koichiro
Published: November 2004

We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three‐step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full‐length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca2+‐binding site. Site‐directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high α‐helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low α‐helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild‐type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild‐types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.
Regulation of connective tissue growth factor (CTGF/CCN2) gene transcription and mRNA stability in smooth muscle cells
Authors: Chowdhury Ibrul , Chaqour Brahim
Published: November 2004

Connective tissue growth factor (CTGF/CCN2) is an immediate early gene‐encoded polypeptide modulating cell growth and collagen synthesis. The importance of CTGF/CCN2 function is highlighted by its disregulation in fibrotic disorders. In this study, we investigated the regulation and signaling pathways that are required for various stimuli of intracellular signaling events to induce the expression of the endogenous CTGF/CCN2 gene in smooth muscle cells. Incubation with the bioactive lysolipid sphingosine 1‐phosphate (S1P) produced a threefold increase, whereas stimulation with either fetal bovine serum or anisomycin induced an even stronger activation (eightfold) of CTGF/CCN2 expression. Using a combination of pathway‐specific inhibitors and mutant forms of signaling molecules, we found that S1P‐ and fetal bovine serum‐induced CTGF/CCN2 expression were dependent on both RhoA GTPase and p38 mitogen‐activated protein kinase transduction pathways, whereas the effects of anisomycin largely involved p38 and phosphatidyl inositol 3‐kinase signaling mechanisms. However, activation via these signaling events was absolutely dependent on actin cytoskeleton integrity. In particular, RhoA‐dependent regulation of the CTGF/CCN2 gene was concomitant to increased polymerization of actin microfilaments resulting in decreased G‐ to F‐actin ratio and appeared to be achieved at the transcriptional level. The p38 signaling pathway was RhoA‐independent and led to CTGF/CCN2 mRNA stabilization. Use of actin‐binding drugs showed that the actual physical state of monomeric G‐actin is a critical determinant for CTGF/CCN2 gene induction. These data indicate that distinct cytoskeletally based signaling events within the intracellular signaling machinery affect either transcriptionally or post‐transcriptionally the expression of the CTGF/CCN2 gene in smooth muscle cells.
Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV‐1 protease inhibitors
Authors: Petroková Hana , Dušková Jarmila , Dohnálek Jan , Skálová Tereza , Vondráčková‐Buchtelová Eva , Souček Milan , Konvalinka Jan , Brynda Jiří , Fábry Milan , Sedláček Juraj , Hašek Jindřich
Published: November 2004

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV‐1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 Å. The peptidomimetic inhibitor Boc‐Phe‐Ψ[CH2CH2NH]‐Phe‐Glu‐Phe‐NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV‐1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV‐1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.