bluuebunny/crossref_metadata_2025 huggingface dataset:
Expansion of Melanoma-specific Cytolytic CD8+ T Cell Precursors in Patients with Metastatic Melanoma Vaccinated with CD34+ Progenitor-derived Dendritic Cells
Authors: Paczesny Sophie , Banchereau Jacques , Wittkowski Knut M. , Saracino Giovanna , Fay Joseph , Palucka A. Karolina
Published: June 2004

Cancer vaccines aim at inducing (a) tumor-specific effector T cells able to reduce/eliminate the tumor mass, and (b) long-lasting tumor-specific memory T cells able to control tumor relapse. We have shown earlier, in 18 human histocompatibility leukocyte antigen (HLA)-A*0201 patients with metastatic melanoma, that vaccination with peptide-loaded CD34–dendritic cells (DCs) leads to expansion of melanoma-specific interferon γ–producing CD8+ T cells in the blood. Here, we show in 9 out of 12 analyzed patients the expansion of cytolytic CD8+ T cell precursors specific for melanoma differentiation antigens. These precursors yield, upon single restimulation with melanoma peptide–pulsed DCs, cytotoxic T lymphocytes (CTLs) able to kill melanoma cells. Melanoma-specific CTLs can be grown in vitro and can be detected in three assays: (a) melanoma tetramer binding, (b) killing of melanoma peptide–pulsed T2 cells, and (c) killing of HLA-A*0201 melanoma cells. The cytolytic activity of expanded CTLs correlates with the frequency of melanoma tetramer binding CD8+ T cells. Thus, CD34-DC vaccines can expand melanoma-specific CTL precursors that can kill melanoma antigen–expressing targets. These results justify the design of larger follow-up studies to assess the immunological and clinical response to peptide-pulsed CD34-DC vaccines.
Low Dose Leishmania major Promotes a Transient T Helper Cell Type 2 Response That Is Down-regulated by Interferon γ–producing CD8+ T Cells
Authors: Uzonna Jude E. , Joyce Karen L. , Scott Phillip
Published: June 2004

An unresolved issue in the field of T helper (Th) cell development relates to the findings that low doses of antigen promote Th2 cell development in vitro, whereas several classic in vivo studies suggest the opposite. Here we resolve this paradox by studying the early immune response in mice after infection with different doses of Leishmania major. We found that low parasite doses induced a Th2 response in C57BL/6 (B6) mice, whereas high doses induced a Th1 response. However, the Th2 response in low dose–infected mice was transient and the animals healed. The appearance of a Th1 response after low dose infection was dependent upon the concomitant activation of interferon γ–producing CD8+ T cells. In the absence of CD8+ T cells, the Th2 response was maintained. However, either neutralization of interleukin (IL)-4 or administration of IL-12 promoted a Th1 response after low dose infection of CD8-deficient mice, indicating that the required role for CD8+ T cells was limited to modulation of CD4+ T cell responses. Thus, the discrepant results seen between in vivo and in vitro studies on the effects of antigen dose on Th cell differentiation may depend upon whether CD8+ T cells participate in the immune response.
CD4+CD25+ T Regulatory Cells Dependent on ICOS Promote Regulation of Effector Cells in the Prediabetic Lesion
Authors: Herman Ann E. , Freeman Gordon J. , Mathis Diane , Benoist Christophe
Published: June 2004

CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69− cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69− cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory–specific profile. Thus, CD4+CD25+69− Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.
p53 Localization at Centrosomes during Mitosis and Postmitotic Checkpoint Are ATM-dependent and Require Serine 15 Phosphorylation
Authors: Tritarelli A. , Oricchio E. , Ciciarello M. , Mangiacasale R. , Palena A. , Lavia P. , Soddu S. , Cundari E.
Published: June 2004

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia–derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint
Do procalcitonin and C-reactive protein levels have a place in the diagnosis and follow-up of Helicobacter pylori infections?
Authors: Saribas Suat , Kocazeybek Bekir , Aslan Mustafa , Altun Sibel , Seyhun Yalcın , Öner Y. Ali , Memisoglu Nejat
Published: June 2004

The aims of this study were to determine the levels of procalcitonin (PCT) and C-reactive protein (CRP) in Helicobacter pylori-positive (HP+) patients diagnosed with duodenal and gastric ulcer and to evaluate the correlation of PCT and CRP levels with other invasive and non-invasive diagnostic methods for determination of H. pylori eradication in post-treatment follow-up. Thirty-five HP+ patients with dyspepsia were included in this study. Serum samples (5 ml) were collected at admission and after 24 h. Antimicrobial therapy (omeprazole, amoxycillin and clarithromycin) was given for 1 week to HP+ patients who were positive only by culture or by urease test plus pathology. After 1 month, serum samples (5 ml) were collected again and culture, urease and pathology investigations were performed on endoscopic samples. PCT and CRP levels were measured in the collected blood samples. Thirty-five H. pylori-negative (HP−) cases with dyspepsia, 38 cases with bacteraemia and 35 healthy blood donors were included in this study as control groups. The mean and minimum–maximum levels of PCT were 1.39 (0.25–6.75), 0.35 (0.12–0.71), 7.45 (0.68–51.5) and 0.40 (0.12–0.71) ng ml−1 for the groups of HP+, HP− and bacteraemia patients and healthy donors, respectively. Mean CRP levels were 1.00 (<0.5–8.11), 0.62 (<0.5–3.2), 11.5 (3.2–43.5) and 0.63 (<0.5–5.46) mg dl−1 for the same groups. A statistically significant difference was found between HP+ patients and both HP− cases and healthy blood donors for PCT levels, and higher PCT levels were found on admission in cases of bacteraemia than in the other groups (P < 0.05). PCT levels of HP+ cases decreased significantly (from 1.39 to 0.86) between admission and the post-treatment period (30 days); however, PCT levels remained higher than the cut-off value (0.5 ng ml−1). Similar ranges of CRP levels were found over the same time-period. The sensitivity of PCT was found to be higher than that of CRP on admission, but the specificity of PCT was found to be lower than that of CRP on the day of admission (65 and 74 %, respectively). The sensitivity of PCT was the same as that of CRP for the post-treatment period, but specificity of PCT was higher than that of CRP for the post-treatment period (83 and 76 %, respectively). It was concluded that PCT and CRP are not very effective markers for H. pylori infection in primary diagnosis or in eradication follow-up after therapy when used in parallel with conventional diagnostic methods, even if there is a difference in PCT and CRP levels between HP+ and HP− cases on admission.
Non-invasive diagnosis of Helicobacter pylori infection in adult dyspeptic patients by stool antigen detection: does the rapid immunochromatography test provide a reliable alternative to conventional ELISA kits?
Authors: Chisholm Stephanie A. , Watson Claire L. , Teare E. Louise , Saverymuttu Seth , Owen Robert J.
Published: June 2004

Stool antigen-testing allows non-invasive detection of Helicobacter pylori that is indicative of active infection. Three commercial kits are currently marketed in the UK for stool antigen-testing. The aim of this study was to conduct a comparative evaluation of the performances of each of these tests, compared with culture and histological examination of gastric biopsies, for pre-treatment diagnosis of infection in an adult dyspeptic population in south-east England. Examination of 112 stool samples by the Premier Platinum HpSA ELISA (Meridian Diagnostics) and by the Amplified IDEIA HpStAR ELISA (DakoCytomation) kits demonstrated that the latter was more sensitive (81.3 versus 93.8 %, respectively) and specific (91.7 versus 100.0 %, respectively). Additionally, the IDEIA HpStAR was easier to interpret, with OD readings of positive and negative results being far from the recommended cut-off, whereas equivocal results that were generated by the HpSA kit were difficult to interpret. Additional testing of 87 of the 112 stools by the ImmunoCard STAT! HpSA kit (Meridian Diagnostics) demonstrated that this test was easier to perform than ELISA and was more sensitive than the HpSA kit but, compared with the IDEIA HpStAR kit, the ImmunoCard test was less sensitive (87.8 versus 95.9 %, respectively) and specific (89.4 versus 100.0 %, respectively). Furthermore, the ImmunoCard test generated weakly positive results, correlating with lower OD readings for both ELISA kits, that were difficult to interpret. The Amplified IDEIA HpStAR kit is therefore the most sensitive and specific of the three tests that are available for pre-treatment, non-invasive detection of H. pylori in stool samples in an English adult dyspeptic population.
Genotypic diversity and virulence traits of Streptococcus mutans in caries-free and caries-active individuals
Authors: Napimoga Marcelo Henrique , Kamiya Regianne Umeko , Rosa Rosimeire Takaki , Rosa Edvaldo AntonioR. , Höfling José Francisco , de Oliveira Mattos-Graner Renata , Gonçalves Reginaldo Bruno
Published: June 2004

The present study evaluated the relationship between clonal diversity and some virulence traits of Streptococcus mutans isolated from eight caries-free and eight caries-active subjects. A total of 155 S. mutans isolates from caries-free subjects and 144 isolates from caries-active subjects were obtained from samples of saliva, dental plaque and tongue surface and identified by PCR. The isolates were submitted to arbitrarily primed (AP)-PCR (OPA-2 and OPA-13) and multilocus enzyme electrophoresis (MLEE) to establish the genotypic diversity. Production of water-insoluble glucan (WIG) (monitored by SDS-PAGE), final pH of cultures and the ability of bacterial cells to adhere to smooth glass in the presence of sucrose were measured. High and comparable abilities of MLEE and AP-PCR were found to distinguish S. mutans genotypes, using Simpson's index of discrimination (0.971 and 0.968, respectively). The results showed a significant difference (P < 0.01) in the number of genotypes when caries-free and caries-active groups were compared by both fingerprinting methods used. Final pH (P = 0.32) and the percentage of adherence to a glass surface (P = 0.62) did not show differences between the two groups; however, the intensities of WIG bands from the caries-active group were greater than those from the caries-free group (P < 0.01). In addition, WIG was positively correlated with the ability of S. mutans to adhere to a glass surface (r = 0.34, P = 0.02) from caries-active subjects. These data showed that AP-PCR analysis and MLEE are both effective methods for assessing the genetic relatedness of S. mutans. Using these techniques, it was found that there is a larger number of genotypes of S. mutans with increased ability to synthesize WIG in caries-active individuals.
Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens
Authors: Flori Pierre , Bellete Bahrie , Durand Fabrice , Raberin Hélène , Cazorla Céline , Hafid Jamal , Lucht Frédéric , Sung Roger Tran Manh
Published: June 2004

Between January 2002 and July 2003, 173 bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100 % for staining (where either one or both techniques were positive), 100 and 87.0 % for conventional PCR and 100 and 84.9 % for real-time PCR, respectively. The use of a concentration of 103 copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84.9 to 98.6 % without reducing the sensitivity of the technique. This technique is rapid (<3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with <103 copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.
Characterization of Neisseria meningitidis isolates collected from 1974 to 2003 in Japan by multilocus sequence typing
Authors: Takahashi Hideyuki , Kuroki Toshiro , Watanabe Yuko , Tanaka Hiroshi , Inouye Hiroo , Yamai Shiro , Watanabe Haruo
Published: June 2004

Analysis of 182 Neisseria meningitidis strains isolated over the past 30 years in Japan by serogroup typing and multilocus sequence typing (MLST) was performed. The serogroups of the 182 Japanese isolates were B (103 isolates), Y (39), W135 (1) and non-groupable (39). By MLST analysis, 65 different sequence types (ST) were identified, 42 of which were not found in the MLST database as of January 2004 and seemed to be unique to Japan. Statistical analysis of the MLST results revealed that, although the Japanese isolates seemed to be genetically divergent, they were classified into six major clonal complexes and other minor complexes. Among these isolates, well-documented ST complexes found worldwide were present, such as ST-23 complex (49 isolates), ST-44 complex (41 isolates) and ST-32 complex (8 isolates). On the other hand, a new clonal complex designated ST-2046 complex (28 isolates), which has not been identified in other countries, was also found, suggesting that this clone was indigenous to Japan. Taken together, it was speculated that meningococcal isolates in Japan comprised heterogeneous clones, which were derived both from clones identified in other countries and clones unique to Japan.
Immunocytochemical studies of Salmonella Typhimurium invasion of porcine jejunal epithelial cells
Authors: Schauser Kirsten , Olsen John Elmerdahl , Larsson Lars-Inge
Published: June 2004

Although infection of pigs with Salmonella Typhimurium represents a serious problem, most studies on Salmonella infection have been carried out in other species. The purpose of the current study was to examine the route(s) of entry of Salmonella Typhimurium in pigs, using a jejunal loop model. The infection process was followed over 240 min using single to triple immunocytochemical detection of Salmonella and intestinal cell markers. Salmonella invasion was observed in both cytokeratin-18-positive and -negative cylindrical absorptive cells within 5–10 min. Subepithelial invasion of ordinary villi was consistently less marked than invasion of the subepithelial layer of Peyer's patches. Our results show that several epithelial cell types were invaded by Salmonella, and that Peyer's patches represent the main portal of entry in early Salmonella infection. Additionally, infection was associated with alterations in the keratin and F-actin cytoskeleton of intestinal epithelial cells, probably reflecting toxin-mediated actions. Such changes were confined to the proximal region of the jejunum, demonstrating a regional heterogeneity of intestinal epithelial cell responses to Salmonella infection.
Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales
Authors: Scott Fiona W. , Pitt Tyrone L.
Published: June 2004

Most past studies of cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients in the UK suggest that it is a rare occurrence. However, two recent reports of highly transmissible strains in patients in regional centres in England (Liverpool and Manchester) have raised questions as to the extent of the problem and prompted a nationwide survey to establish the distribution of P. aeruginosa strain genotypes among these patients. Isolates of P. aeruginosa were requested from over 120 hospitals in England and Wales and a sample size of approximately 20 % of the CF patient population in each centre was recommended. In total, 1225 isolates were received from 31 centres (range 1 to 330). Single patient isolates were typed by SpeI macrorestriction and PFGE. A panel of strains of the common genotypes including representatives of reported transmissible strains was assembled and further characterized by fluorescent amplified fragment length polymorphism (FAFLP) genotyping. At least 72 % of all patients harboured strains with unique genotypes. Small clusters of related strains were evident in some centres, presumably indicating limited transmission of local strains. The most prevalent strain was indistinguishable from that previously described as the ‘Liverpool’ genotype, and accounted for approximately 11 % of patient isolates from 15 centres in England and Wales. The second most common genotype (termed Midlands 1) was recovered from 86 patients in nine centres and the third genotype, which matched closely the PFGE profile of Clone C, a genotype originally described in Germany, was found in eight centres and was isolated from 15 patients. A fourth genotype, identical to the published Manchester strain, was found in three centres. FAFLP analysis revealed some microheterogeneity among strains of the Liverpool genotype but all isolates of this genotype were positive by PCR for a strain-specific marker. These data suggest that cross-infection with P. aeruginosa has occurred both within and widely between CF centres in England and Wales. The two most common genotypes accounted for more than one-fifth of patients’ isolates examined and transmissible genotypes were found in all but three centres studied. These results emphasize the need for continued surveillance of P. aeruginosa genotypes in CF patients to provide informed infection control policy in treatment centres.
The Campylobacter jejuni general glycosylation system is important for attachment to human epithelial cells and in the colonization of chicks
Authors: Karlyshev A. V. , Everest P. , Linton D. , Cawthraw S. , Newell D. G. , Wren B. W.
Published: June 2004

It has recently been shown that the enteropathogenCampylobacter jejunihas anN-linked generalproteinglycosylation pathway (Pgl) that modifies many of the organism's proteins. To determine the role of theN-linked general glycosylation inC jejuni, the authors studied thepglHgene, which shows high similarity to a family of sugar transferases.pglHmutants were constructed in strains 81116 and 11168H. Both mutants were shown to be deficient in their ability to glycosylate a number ofC. jejuniproteins, but their lipooligosaccharide and capsule were unaffected. ThepglHmutants had significantly reduced ability to adhere to and invade human epithelial Caco-2 cells. Additionally, the 81116pglHmutant was severely affected in its ability to colonize chicks. These results suggest that glycosylation is important for the attachment ofC. jejunito human and chicken host cells and imply a role for glycoproteins in the pathogenesis ofC. jejuni.
Diversity analysis of commensal porcine Escherichia coli – associations between genotypes and habitat in the porcine gastrointestinal tract
Authors: Dixit Sameer M. , Gordon David M. , Wu Xi-Yang , Chapman Toni , Kailasapathy Kaila , Chin James J.-C.
Published: June 2004

Diversity studies of entericEscherichia colihave relied almost entirely on faecal isolations on the assumption that they are representative of flora found throughout the gastrointestinal tract. The authors have addressed this belief by analysing isolates obtained from the duodenum, ileum, colon and faeces of pigs.E. coliisolates were obtained from eight pigs and characterized using multi-locus enzyme electrophoresis and PCR-based screening for a range of factors thought to be associated with intestinal and extra-intestinal disease. There are four main genetic groups of commensalE. coli(A, B1, B2, D). Group A strains represented 76 % of the isolates from the duodenum, ileum and colon compared to 58 % of the strains isolated from faeces. A nested molecular analysis of variance based on the allozyme and virulence factor screening results showed that differences among individual pigs accounted for 6 % of the observed genetic diversity, whilst 27 % of the genetic variation could be explained by clonal composition differences among gut regions. Finally, the absence of virulence genes in these commensals indicates that they may be suitable as a probiotic consortium, particularly if they also display increased adherence to enterocytes and antagonistic activity against pathogenic strains ofE. coli.
Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent
Authors: Liefting Lia W. , Shaw Mary E. , Kirkpatrick Bruce C.
Published: June 2004

The complete nucleotide sequences of the two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent (BLTVA) have been determined. The larger plasmid, pBLTVA-1, was 10 785 nt in length and contained 11 putative ORFs, almost all of which were duplicated or triplicated on the plasmid due to the presence of large repeated regions. The sequence contained a series of tandem repeats, the largest of which was 338 nt long. The sequences of ORFs 4 and 11 showed homology with the replication genes of plasmids from other phytoplasmas and from geminiviruses. ORF9, the only ORF present as a single copy, showed homology with DNA primase genes from bacterial chromosomes and contained the conserved zinc finger and topoisomerase/primase domains. None of the other eight ORFs showed homology with known sequences in the GenBank database. pBLTVA-2 was 2587 nt in length, and all of its sequence was nearly identical to sequences from pBLTVA-1, most of which spanned ORFs 10 and 11, including the 338 nt tandem repeat. Analysis of 30 strains of BLTVA showed that most of the 11 putative ORFs were present, but the size of the plasmids varied in these strains.
Substrate recognition by nonribosomal peptide synthetase multi-enzymes
Authors: Lautru Sylvie , Challis Gregory L.
Published: June 2004

Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is less clear for the thioesterase domains, which seem in certain cases to be dedicated to the hydrolysis/cyclization of their natural substrate, whereas in other cases they are largely permissive.
Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation
Authors: Shleeva Margarita , Mukamolova Galina V. , Young Michael , Williams Huw D. , Kaprelyants Arseny S.
Published: June 2004

Conditions were investigated that promote the formation of ‘non-culturable’ (NC) cells ofMycobacterium(Myc.)smegmatisin stationary phase. After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified Hartman's–de Bont medium), bacteria adopted a stable NC state after 3–4 days incubation in stationary phase. Such conditions are not specific aspurFanddevRmutants ofMyc. smegmatisalso showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's–de Bont medium) was similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway. NC cells of wild-typeMyc. smegmatisresume growth when transferred to a suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium. Moreover, co-culture withMicrococcus(Mcc.)luteuscells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains ofMyc. smegmatisharbouring plasmids containing theMcc. luteus rpfgene also adopt a similar NC state after growth to stationary phase in modified Hartman's–de Bont medium. However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis ofMcc. luteusRpf. Resuscitation was not observed in the control strain harbouring a plasmid lackingMcc. luteus rpf. In contrast to wild-type, the NC cells ofpurFanddevRmutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.
Mutant alleles of the essential 14-3-3 gene in Candida albicans distinguish between growth and filamentation
Authors: Palmer Glen E. , Johnson Kevin J. , Ghosh Sumana , Sturtevant Joy
Published: June 2004

The opportunistic fungal pathogenCandida albicanshas the ability to exploit diverse host environments and can either reside commensally or cause disease. In order to adapt to its new environment it must respond to new physical conditions, nutrient sources, and the host immune response. This requires the co-regulation of multiple signalling networks. The 14-3-3 family of proteins is highly conserved in all eukaryotic species. These proteins regulate signalling pathways involved in cell survival, the cell cycle, and differentiation, and effect their functions via interactions with phosphorylated serines/threonines. InC. albicansthere is only one 14-3-3 protein, Bmh1p, and it is required for vegetative growth and optimal filamentation. In order to dissect separate functions of Bmh1p inC. albicans, site-directed nucleotide substitutions were made in theC. albicans BMH1gene based on studies in other species. Putative temperature-sensitive, ligand-binding and dimerization mutants were constructed. In addition two mutant strains identified through random mutagenesis were analysed. All five mutant strains demonstrated varying defects in growth and filamentation. This paper begins to segregate functions of Bmh1p that are required for optimal growth and the different filamentation pathways. These mutant strains will allow the identification of 14-3-3 target interactions and correlate the individual functions of Bmh1p to cellular processes involved in pathogenesis.
The hyaluronate lyase of Staphylococcus aureus – a virulence factor?
Authors: Makris George , Wright John D. , Ingham Eileen , Holland Keith T.
Published: June 2004

The hyaluronate lyase (HL) gene ofStaphylococcus aureus8325-4 (hysA) was inactivatedin vitrowith the insertion of the erythromycin determinant,ermC, from plasmid pE194. ThehysA : : ermCmutation was introduced intoS. aureusvia a temperature-sensitive shuttle vector, where it underwent homologous recombination with the wild-type (w.t.) allele. The insertion ofermCin the chromosomalhysAlocus was confirmed by Southern blot hybridization and the loss of HL activity was demonstrated macroscopically by a plate assay. The importance of HL for pathogenicity was assessed by comparing the virulence of the HL−mutant strain to that of the w.t. in an established mouse abscess model ofS. aureusinfection. A significantly higher cell recovery was obtained from lesions infected with the w.t. strain compared to the lesions infected with the HL−strain (P=0·01). Although the lesion areas from both groups were not significantly different (P=0·9) they were of different morphology. A colorimetric assay was used to measure HL activity from culture supernatants of theS. aureus8325-4 strains w.t., WA250 (agr) and PC1839 (sar) grown in a chemically defined medium. HL activity reached a maximum in the w.t. strain during mid-exponential phase (t=5 h) and while it showed a 16-fold decrease in theagrmutant it increased 35-fold in thesarmutant background. These results strongly suggest that HL is a virulence factor which is important in the early stages of subcutaneous infections.
The role of dor gene products in controlling the P2 promoter of the cytochrome c 2 gene, cycA, in Rhodobacter sphaeroides
Authors: Tavano Christine L. , Comolli James C. , Donohue Timothy J.
Published: June 2004

This study explores the regulatory networks controlling anaerobic energy production by the facultative phototrophRhodobacter sphaeroides. The specific aim was to determine why activity of the P2 promoter for the gene (cycA) encoding the essential photosynthetic electron carrier, cytochromec2, is decreased when the alternative electron acceptor DMSO is added to photosynthetically grown cells. The presence of DMSO is believed to activate the DorR response regulator, which controls expression of proteins required to reduce DMSO. A DorR−strain showed no change incycAP2 promoter activity when DMSO was added to photosynthetic cells, indicating that DorR was required for the decreased expression in wild-type cells. To test if DorR acted directly at this promoter to change gene expression, recombinant DorR was purified and studiedin vitro. Preparations of DorR that were active at other target promoters showed no detectable interaction withcycAP2, suggesting that this protein is not a direct regulator of this promoter. We also found thatcycAP2 activity in a DorA−strain was not decreased by the addition of DMSO to photosynthetic cells. A model is presented to explain why the presence of a functional DMSO reductase (DorA) is required for DMSO to decreasecycAP2 expression under photosynthetic conditions.
Multiple linear regression analysis of bacterial deposition to polyurethane coatings after conditioning film formation in the marine environment
Authors: Bakker Dewi P. , Busscher Henk J. , van Zanten Joyce , de Vries Joop , Klijnstra Job W. , van der Mei Henny C.
Published: June 2004

Many studies have shown relationships of substratum hydrophobicity, charge or roughness with bacterial adhesion, although bacterial adhesion is governed by interplay of different physico-chemical properties and multiple regression analysis would be more suitable to reveal mechanisms of bacterial adhesion. The formation of a conditioning film of organic compounds adsorbed from seawater affects the properties of substratum surfaces prior to bacterial adhesion, which is a complicating factor in studying the mechanism of bacterial adhesion. In this paper, the impact of conditioning films adsorbed from natural seawater to four polyurethane coatings with different hydrophobicity, elasticity and roughness was studied for three different marine bacterial strains in a multiple linear regression analysis. The water contact angle on hydrophobic coatings decreased on average by 8 degrees and increased on average by the same amount on hydrophilic coatings. These changes were accompanied by increased concentrations of oxygen and nitrogen on the surface as determined by X-ray photoelectron spectroscopy, indicative of adsorption of proteinaceous material. Furthermore, the mean surface roughness increased on average by 4 nm after conditioning film formation. Multiple linear regression analysis revealed that changes in deposition due to conditioning film formation ofMarinobacter hydrocarbonoclasticus,Psychrobactersp. SW5H andHalomonas pacificain a stagnation-point flow chamber could be explained in a model comprising hydrophobicity and the prevalence of nitrogen-rich components on the surface for the most hydrophobic strain. For the two more hydrophilic strains, deposition was governed by a combination of surface roughness and hydrophobicity. Elasticity was not a factor in bacterial adhesion to conditioning films.
Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans
Authors: Oka Takuji , Hamaguchi Tetsu , Sameshima Yuka , Goto Masatoshi , Furukawa Kensuke
Published: June 2004

ProteinO-glycosylation is essential for protein modification and plays important roles in eukaryotic cells.O-Mannosylation of proteins occurs in the filamentous fungusAspergillus. The structure and function of thepmtAgene, encoding proteinO-d-mannosyltransferase, which is responsible for the initialO-mannosylation reaction inAspergillus nidulans, was characterized. Disruption of thepmtAgene resulted in the reduction ofin vitroproteinO-d-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. ThepmtAdisruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall inA. nidulans.
Oxidative stress response in Clostridium perfringens
Authors: Jean Delphine , Briolat Valérie , Reysset Gilles
Published: June 2004

Clostridium perfringens, a strictly anaerobic bacterium, is able to survive when exposed to oxygen for short periods of time and exhibits a complex adaptive response to reactive oxygen species, both under aerobic and anaerobic conditions. However, this adaptive response is not completely understood.C. perfringenspossesses specialized genes that might be involved in this adaptive process, such as those encoding superoxide dismutase (SOD), superoxide reductase and alkyl hydroperoxide reductase, but their contribution to the oxidative stress response and their control mechanisms are unknown. By a combination of functional complementation ofEscherichia colistrains impaired in either SOD, alkyl hydroperoxide reductase (AhpC) or catalase activity (Cat), transcription analysis and characterization of mutants impaired in regulatory genes, it was concluded that: (i) the product of thesodgene is certainly essential to scavenge superoxide radicals, (ii) theahpCgene, which is fully induced in all oxidative stress conditions, is probably involved in the scavenging of all intracellular peroxides, (iii) the three rubrerythrin (rbr) genes ofC. perfringensdo not encode proteins within vivoH2O2reductase activity, and (iv) the two rubredoxin (rub) genes do not contribute to the hypothetical superoxide reductase activity, but are likely to belong to an electron transfer chain involved in energy metabolism.
Phototrophic utilization of taurine by the purple nonsulfur bacteria Rhodopseudomonas palustris and Rhodobacter sphaeroides
Authors: Novak Ryan T. , Gritzer Rachel F. , Leadbetter Edward R. , Godchaux Walter
Published: June 2004

Taurine metabolism by two phototrophically grown purple nonsulfur bacteria enrichment isolates has been examined.Rhodopseudomonas palustris(strain Tau1) grows with taurine as a sole electron donor, sulfur and nitrogen source during photoautotrophic growth.Rhodobacter sphaeroides(strain Tau3) grows on the compound as sole electron donor, sulfur and nitrogen source, and partial carbon source, in the presence of CO2during photoheterotrophic growth. Both organisms utilize an inducible taurine–pyruvate aminotransferase and a sulfoacetaldehyde acetyltransferase. The products of this metabolism are bisulfite and acetyl phosphate. Bisulfite ultimately was oxidized to sulfate, but this was not an adequate source of electrons for photometabolism. Experiments using either [U-14C]taurine or14CO2demonstrated thatRb. sphaeroidesTau3 assimilated the carbon from approximately equimolar amounts of taurine and exogenous CO2. The taurine-carbon assimilation was not diminished by excess non-radioactive bicarbonate. Malate synthase (but not isocitrate lyase) was induced in these taurine-grown cells. It is concluded that assimilation of taurine carbon occurs through an intermediate other than CO2. Similar labelling experiments withRp. palustrisTau1 determined that taurine is utilized only as an electron donor for the reduction of CO2, which contributes all the cell carbon. Photoautotrophic metabolism was confirmed in this organism by the absence of either malate synthase or isocitrate lyase in taurine+CO2-grown cells. Culture collection strains of these two bacteria did not utilize taurine in these fashions.
Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae
Authors: Srikumar Ramakrishnan , Mikael Leonie G. , Pawelek Peter D. , Khamessan Ali , Gibbs Bernard F. , Jacques Mario , Coulton James W.
Published: June 2004

From the porcine pathogenActinobacillus pleuropneumoniaecultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of ∼105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology tohgbAofPasteurella multocida. Upon screening two genomic libraries ofA. pleuropneumoniaeserotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region ofhgbAfromA. pleuropneumoniaeserotype 1 showed consensus for −35 and −10 sequences and a putative Fur-binding site. RT-PCR confirmed thathgbAofA. pleuropneumoniaeis upregulated in response to diminished levels of iron in the culture medium. While an internally deletedhgbAmutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth ofA. pleuropneumoniaein the presence of Hb as sole iron source.
Rhodococcus opacus expresses the xsc gene to utilize taurine as a carbon source or as a nitrogen source but not as a sulfur source
Authors: Denger Karin , Ruff Jürgen , Schleheck David , Cook Alasdair M.
Published: June 2004

The Gram-positive bacteria Rhodococcus opacus ISO-5 and Rhodococcus sp. RHA1 utilized taurine (2-aminoethanesulfonate) as the sole source of carbon or of nitrogen or of sulfur for growth. Different gene clusters and enzymes were active under these different metabolic situations. Under carbon- or nitrogen-limited conditions three enzymes were induced, though to different levels: taurine-pyruvate aminotransferase (Tpa), alanine dehydrogenase (Ald) and sulfoacetaldehyde acetyltransferase (Xsc). The specific activities of these enzymes in R. opacus ISO-5 were sufficient to explain the growth rates under the different conditions. These three enzymes were purified and characterized, and the nature of each reaction was confirmed. Analyses of the genome of Rhodococcus sp. RHA1 revealed a gene cluster, tauR-ald-tpa, putatively encoding regulation and oxidation of taurine, located 20 kbp from the xsc gene and separate from two candidate phosphotransacetylase (pta) genes, as well as many candidate ABC transporters (tauBC). PCR primers allowed the amplification and sequencing of the tauR-ald-tpa gene cluster and the xsc gene in R. opacus ISO-5. The N-terminal sequences of the three tested proteins matched the derived amino acid sequences of the corresponding genes. The sequences of the four genes found in each Rhodococcus strain shared high degrees of identity (>95 % identical positions). RT-PCR studies proved transcription of the xsc gene when taurine was the source of carbon or of nitrogen. Under sulfur-limited conditions no xsc mRNA was generated and no Xsc was detected. Taurine dioxygenase (TauD), the enzyme catalysing the anticipated desulfonative reaction when taurine sulfur is assimilated, was presumed to be present because oxygen-dependent taurine disappearance was demonstrated with taurine-grown cells only. A putative tauD gene (with three other candidates) was detected in strain ISO-5. Regulation of the different forms of metabolism of taurine remains to be elucidated.
Naturally occurring horizontal gene transfer and homologous recombination in Mycobacterium
Authors: Krzywinska Elzbieta , Krzywinski Jaroslaw , Schorey Jeffrey S.
Published: June 2004

Acquisition of genetic information through horizontal gene transfer (HGT) is an important evolutionary process by which micro-organisms gain novel phenotypic characteristics. In pathogenic bacteria, for example, it facilitates maintenance and enhancement of virulence and spread of drug resistance. In the genusMycobacterium, to which several primary human pathogens belong, HGT has not been clearly demonstrated. The few existing reports suggesting this process are based on circumstantial evidence of similarity of sequences found in distantly related species. Here, direct evidence of HGT between strains ofMycobacterium aviumrepresenting two different serotypes is presented. Conflicting evolutionary histories of genes encoding elements of the glycopeptidolipid (GPL) biosynthesis pathway led to an analysis of the GPL cluster genomic sequences from fourMycobacterium aviumstrains. The sequence ofM. aviumstrain 2151 appeared to be a mosaic consisting of three regions having alternating identities to eitherM. aviumstrains 724 or 104. Maximum-likelihood estimation of two breakpoints allowed a ∼4100 bp region horizontally transferred into the strain 2151 genome to be pinpointed with confidence. The maintenance of sequence continuity at both breakpoints and the lack of insertional elements at these sites strongly suggest that the integration of foreign DNA occurred by homologous recombination. To our knowledge, this is the first report to demonstrate naturally occurring homologous recombination inMycobacterium. This previously undiscovered mechanism of genetic exchange may have major implications for the understanding ofMycobacteriumpathogenesis.
Estimating Mean Response as a Function of Treatment Duration in an Observational Study, Where Duration May Be Informatively Censored
Authors: Johnson Brent A. , Tsiatis Anastasios A.
Published: June 2004

Summary. After a treatment is found to be effective in a clinical study, attention often focuses on the effect of treatment duration on outcome. Such an analysis facilitates recommendations on the most beneficial treatment duration. In many studies, the treatment duration, within certain limits, is left to the discretion of the investigators. It is often the case that treatment must be terminated prematurely due to an adverse event, in which case a recommended treatment duration is part of a policy that treats patients for a specified length of time or until a treatment‐censoring event occurs, whichever comes first. Evaluating mean response for a particular treatment‐duration policy from observational data is difficult due to censoring and the fact that it may not be reasonable to assume patients are prognostically similar across all treatment strategies. We propose an estimator for mean response as a function of treatment‐duration policy under these conditions. The method uses potential outcomes and embodies assumptions that allow consistent estimation of the mean response. The estimator is evaluated through simulation studies and demonstrated by application to the ESPRIT infusion trial coordinated at Duke University Medical Center.
Erosion of Regression Effect in a Survival Study
Authors: O'Quigley John , Natarajan Loki
Published: June 2004

Summary. Lack of persistence, or erosion, of the regression effect is an alternative to proportional hazards of particular interest in many medical applications. Such a departure from proportional hazards is often the most likely direction in which the model may be inadequate. Questions such as, is the effect of treatment only transitory or to what extent does an initially measured prognostic variable maintain its impact, frequently arise. In the context of a simple changepoint model, we propose a test of the null hypothesis of proportional hazards against the specific alternative of erosion of the regression effect. The particular changepoint model used can be viewed as a first approximation to a more complex reality, an approximation that enables us to avoid specifically modeling the functional form that any erosion might take. Practical guidelines for carrying out the test are provided. The approach is illustrated in the context of a study on risk factors for breast cancer survival.