bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach
Authors: Cox Robert A.
Published: May 2004

Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 °C, and Escherichia coli B/r, grown at 37 °C. A frame of reference was established based on quantitative relationships observed between specific growth rates (μ) of cells and their macromolecular compositions. The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate. It was inferred that the ultra-fast growth of E. coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis. The maximum growth rate of E. coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy. Three characteristic properties of S. coelicolor A3(2) growing optimally (μ=0·30 h−1) were identified. First, the rate of DNA replication was found to approach the rate reported for E. coli (μ=1·73 h−1); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E. coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu. An equation was derived for E. coli B/r relating μ to the number of rrn operons per genome. Values of μ=0·69 h−1 and μ=1·00 h−1 were obtained respectively for cells with one or two rrn operons per genome. Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M. tuberculosis with one rrn operon should be capable of growing much faster than it actually does. Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.
NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria
Authors: Dedysh Svetlana N. , Ricke Peter , Liesack Werner
Published: May 2004

The ability to utilize dinitrogen as a nitrogen source is an important phenotypic trait in most currently known methanotrophic bacteria (MB). This trait is especially important for acidophilic MB, which inhabit acidic oligotrophic environments, highly depleted in available nitrogen compounds. Phylogenetically, acidophilic MB are most closely related to heterotrophic dinitrogen-fixing bacteria of the genusBeijerinckia. To further explore the phylogenetic linkage between these metabolically different organisms, the sequences ofnifHandnifDgene fragments from acidophilic MB of the generaMethylocellaandMethylocapsa, and from representatives ofBeijerinckia, were determined. For reference,nifHandnifDsequences were also obtained from some type II MB of the alphaproteobacterialMethylosinus/Methylocystisgroup and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences ofnifHandnifDwere highly congruent. The phylogenetic relationships among MB in the NifH and NifD trees also agreed well with the corresponding 16S rRNA-based phylogeny, except for two distinctive features. First, different methods used for phylogenetic analysis grouped the NifH and NifD sequences of strains of the gammaproteobacterial MBMethylococcus capsulatuswithin a clade mainly characterized byAlphaproteobacteria, including acidophilic MB and type II MB of theMethylosinus/Methylocystisgroup. From this and other genomic data fromMethylococcus capsulatusBath, it is proposed that an ancient event of lateral gene transfer was responsible for this aberrant branching. Second, the identity values of NifH and NifD sequences betweenMethylocapsa acidiphilaB2 and representatives ofBeijerinckiawere clearly higher (98·5 and 96·6 %, respectively) than would be expected from their 16S rRNA-based relationships. Possibly, these two bacteria originated from a common acidophilic dinitrogen-fixing ancestor, and were subject to similar evolutionary pressure with regard to nitrogen acquisition. This interpretation is corroborated by the observation that, in contrast to most other diazotrophs,M. acidiphilaB2 andBeijerinckiaspp. are capable of active growth on nitrogen-free media under fully aerobic conditions.
Comparison of the structure–activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features
Authors: Collis Christina M. , Hall Ruth M.
Published: May 2004

Incorporation of gene cassettes into integrons occurs by IntI-mediated site-specific recombination between a 59-base element (59-be) site in the cassette and an attI site in the integron. While the 59-be sites share common features and are recognized by several different IntI recombinases, the sequences of attI sites are not obviously related and are preferentially recognized by the cognate IntI. To determine the features of attI sites that are required for recombination proficiency, the structure–activity relationships of a second attI site, the attI3 site from the class 3 integron, were examined. The attI3 site was confined to within a region consisting of 68 bp from the integron backbone and 15 bp from the adjacent cassette. This region includes four IntI3-binding sites, as assessed by gel shift and methylation interference studies. Two of the binding sites are inversely oriented and constitute a simple site that includes the recombination crossover point. The two additional binding sites appear to be directly oriented and one of them is essential for efficient recombination of the attI3 site with a 59-be, but not for recombination with a second full-length attI3 site, which occurs at 100-fold lower frequency. The fourth site enhances attI3 with 59-be recombination 10-fold. The finding that the organization and overall properties of attI3 are very similar to those of attI1 indicates that these features are likely to be common to all attI sites.
Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans
Authors: Frota Cristiane C. , Hunt Debbie M. , Buxton Roger S. , Rickman Lisa , Hinds Jason , Kremer Kristin , van Soolingen Dick , Colston M. Joseph
Published: May 2004

Mycobacterium microti, a member of theMycobacterium tuberculosiscomplex, is phylogenetically closely related toM. tuberculosis, differing in a few biochemical properties. However, these species have different levels of virulence in different hosts; most notablyM. microtishows lower virulence for humans thanM. tuberculosis. This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity ofM. microtistrains. Compared toM. tuberculosisH37Rv, 13 deletions were identified in 12 strains ofM. microti, including the regions RD1 to RD10, which are also missing inMycobacterium bovisBCG. In addition, four new deleted regions, named MiD1, RD1β, MiD2 and MiD3, were identified. DNA sequencing was used to define the extent of most of the deletions in one strain. Although RD1 ofM. bovisBCG andM. microtiis thought to be crucial for attenuation, in this study, three of the fourM. microtistrains that were isolated from immunocompetent patients had the RD1 deletion. In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all theM. microtistrains. These deletions might therefore have some relation to the different host range ofM. microti. It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source. Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion inM. microti.
Enhanced motility of a Proteus mirabilis strain expressing hybrid FlaAB flagella
Authors: Manos Jim , Artimovich Elena , Belas Robert
Published: May 2004

Proteus mirabilishas two tandemly arranged flagellin-encoding genes,flaAandflaB.flaAis transcribed from aσ28promoter, whileflaBis silent.flaAandflaBcan undergo reversible rearrangement to produce a set of hybrid genes referred to asflaAB. Flagellins composed of FlaAB protein have a different amino acid sequence and are antigenically distinct from flagellin composed of FlaA, implicating flagellin gene conversion as a putative virulence mechanism forP. mirabilis. The change in amino acid sequence is also hypothesized to alter the filament helix and, hence, affect the motility of FlaAB-expressing strains. To test this hypothesis, the motility of wild-typeP. mirabiliswas compared with that of a strain, DF1003, locked into the FlaAB+hybrid phase, under conditions of altered ionic strength, pH and viscosity. Cell motion tracking analysis showed that DF1003 has wild-type swimming velocity at physiological conditions, but moves significantly faster and travels further compared to the wild-type at NaCl concentrations greater than 170 mM. DF1003 is also significantly faster than the wild-type at pH 5·2, 5·8 and 8·2, and at 5 and 10 % polyvinylpyrrolidone. Measurements of amplitude and wavelength for isolated flagella subjected to pH 5·8 or 425 mM NaCl showed a loss of helical structure in FlaA flagella compared to FlaAB filaments, a feature that could significantly affect motility under these conditions. These results support a hypothesis that FlaAB flagellin imparts a motile advantage toP. mirabilisin conditions that otherwise may impede bacterial movement. In a broader context, flagellar antigenic variation, commonly thought to serve as means to avoid host defences, may also enhance motility in other bacterial species, thus aiding in the adaptation and survival of the cells.
Induction of Mycobacterium avium growth restriction and inhibition of phagosome–endosome interactions during macrophage activation and apoptosis induction by picolinic acid plus IFNγ
Authors: Pais Teresa F. , Appelberg Rui
Published: May 2004

Treatment of mouse macrophages with picolinic acid (PA) andγ-interferon (IFNγ) led to the restriction ofMycobacterium aviumproliferation concomitant with the sequential acquisition of metabolic changes typical of apoptosis, mitochondrial depolarization, annexin V staining and caspase activation, over a period of up to 5 days. However, triggering of cell death by ATP, staurosporine or H2O2failed to affect mycobacterial viability. In contrast to untreated macrophages where extensive interactions between phagosomes and endosomes were observed, phagosomes from treated macrophages lost the ability to acquire endosomal dextran.N-Acetylcysteine was able to revert both the anti-mycobacterial activity of treated macrophages as well as the block in phagosome–endosome interactions. The treatment, however, induced only a minor increase in the acquisition of lysosomal markers, namely Lamp-1, and did not increase to any great extent the acidification of the phagosomes. These data thus suggest that the anti-mycobacterial activity of PA and IFNγdepends on the interruption of intracellular vesicular trafficking, namely the blocking of acquisition of endosomal material by the microbe.
Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients
Authors: Kus Julianne V. , Tullis Elizabeth , Cvitkovitch Dennis G. , Burrows Lori L.
Published: May 2004

Type IV pili (TFP) are important colonization factors of the opportunistic pathogenPseudomonas aeruginosa, involved in biofilm formation and attachment to host cells. This study undertook a comprehensive analysis of TFP alleles in more than 290 environmental, clinical, rectal and cystic fibrosis (CF) isolates ofP. aeruginosa. Based on the results, a new system of nomenclature is proposed, in whichP. aeruginosaTFP are divided into five distinct phylogenetic groups. Each pilin allele is stringently associated with characteristic, distinct accessory genes that allow the identification of the allele by specific PCR. The invariant association of the pilin and accessory genes implies horizontal transfer of the entire locus. Analysis of pilin allele distribution among isolates from various sources revealed a striking bias in the prevalence of isolates with group I pilin genes from CF compared with non-CF human sources (P<0·0001), suggesting this particular pilin type, which can be post-translationally modified by glycosylation via the action of TfpO (PilO), may confer a colonization or persistence advantage in the CF host. This allele was also predominant in paediatric CF isolates (29 of 43; 67·4 %), showing that this bias is apparent early in colonization. Group I pilins were also the most common type found in environmental isolates tested. To the authors' knowledge, this is the first example of aP. aeruginosavirulence factor allele that is strongly associated with CF isolates.
Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum
Authors: Millership Jason J. , Cai Xiaomin , Zhu Guan
Published: May 2004

Replication protein A (RPA) is a heterotrimeric complex of single-stranded DNA-binding proteins that play multiple roles in eukaryotic DNA metabolism. The RPA complex is typically composed of heterologous proteins (termed RPA1, RPA2 and RPA3) in animals, plants and fungi, which possess different functions. Previously, two distinct, short-type RPA large subunits (CpRPA1 and CpRPA1B) from the apicomplexan parasiteCryptosporidium parvumwere characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although theCpRPA2gene encodes a predicted 40·1 kDa peptide, which is larger than other RPA2 subunits characterized to date, Western blot analysis of oocyst preparations detected a native CpRPA2 protein with a molecular mass of approximately 32 kDa, suggesting that CpRPA2 might undergo post-translational cleavage or the gene was translated at an alternative start codon. Immunofluorescence microscopy using a rabbit anti-CpRPA2 antibody revealed that CpRPA2 protein was mainly distributed in the cytosol (rather than the nuclei) ofC. parvumsporozoites. Semi-quantitative RT-PCR data indicated that CpRPA2 was differentially expressed in a tissue culture model with highest expression in intracellular parasites infecting HCT-8 cells for 36 and 60 h. Sequence comparison suggests that RPA2 is a group of poorly conserved proteins. Nonetheless, functional analyses of recombinant proteins confirmed that CpRPA2 is a single-stranded DNA-binding protein and that it could serve as anin vitrophosphorylation target by a DNA-dependent protein kinase. The minimal length of poly(dT) required for CpRPA2 binding is 17 nucleotides, and the DNA-binding capability was inhibited by phosphorylationin vitro. These observations provide additional evidence on the divergence of RPA proteins betweenC. parvumand host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.
Menadione kills trophozoites and cysts of Giardia intestinalis
Authors: Paget Timothy , Maroulis Sarah , Mitchell Andrew , Edwards Michael R. , Jarroll Edward L. , Lloyd David
Published: May 2004

Production of reactive oxygen species by redox cycling in the presence of low levels of oxygen has been studied as a possible approach to anti-protozoal chemotherapeutic strategy. Incubation of the diplomonad flagellateGiardia intestinaliswith 2-methy-1,4-naphthoquinone (menadione), under anaerobic conditions, gave UV absorption changes characteristic of reduction to menadiol; partial reversal was observed on admitting O2. Under microaerobic conditions, similar to those on the surface of the jejunal mucosa, trophozoites consumed O2rapidly in the presence of menadione; reaction products included singlet O2(monitored by single photon counting of O2-dependent low-level chemiluminescence) and H2O2(measured by the formation of Complex I of microperoxidase). Trophozoites became swollen and incapable of regulatory volume control; these irreversible responses led to loss of motility, cessation of flagellar activity and cell death. Comparison of the sensitivities of trophozoites to metronidazole and menadione gave LC50values (μg ml−1) of 1·2 and 0·7, respectively; corresponding values for cysts (measured byin vitroexcystation capacities) were >50 and 1·3. Menadione (LD50in mice, 0·5 g kg−1) is therefore a potentially more useful and general anti-giardial agent than metronidazole, as it is active against cysts as well as trophozoites.
Spore morphotypes of Thelohania solenopsae (microsporidia) described microscopically and confirmed by PCR of individual spores microdissected from smears by position ablative laser microbeam microscopy
Authors: Sokolova Yuliya Y. , McNally Lacey R. , Fuxa James R. , Vinson S. Bradleigh
Published: May 2004

Development of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8–20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.
Unique organization and regulation of the mrx fimbrial operon in Xenorhabdus nematophila
Authors: He Hongjun , Snyder Holly A. , Forst Steven
Published: May 2004

Xenorhabdus nematophila, a Gram-negative bacterium belonging to the Proteus clade of the family Enterobacteriaceae, forms a mutualistic association with the soil nematode Steinernema carpocapsae. The nematode invades insects and releases Xenorhabdus into the haemolymph, where it participates in insect killing. To begin to understand the role of fimbriae in the unique life cycle of Xenorhabdus, the organization and expression of the mrx fimbrial operon was analysed. The mrx operon contained only five structural genes (mrxACDGH), making it one of the smallest chaperone-usher fimbrial operons studied to date. Unlike the mrp operon of Proteus mirabilis, a site-specific recombinase was not linked to the mrx operon. The intergenic region between the major fimbrial gene (mrxA) and the usher gene (mrxC) lacked a mrpB-like gene, but contained three tandem inverted repeat sequences located downstream of mrxA. A 940 nt mrxA-containing mRNA was the major transcript produced in cells growing on agar, while an mrx polycistronic mRNA was produced at low levels. A canonical σ
70 promoter, identified upstream of mrxA, was not subject to promoter inversion. Fimbriae were not produced in an lrp-mutant strain, suggesting that the leucine-responsive regulatory protein, Lrp, plays a role in the regulation of the mrx operon. These findings show that the genetic organization and regulation of the mrx operon is in several respects distinct from other chaperone-usher fimbrial operons.
Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes
Authors: Arous Safia , Buchrieser Carmen , Folio Patrice , Glaser Philippe , Namane Abdelkader , Hébraud Michel , Héchard Yann
Published: May 2004

The role of the alternativeσ54factor, encoded by therpoNgene, was investigated inListeria monocytogenesby comparing the global gene expression of the wild-type EGDe strain and anrpoNmutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in therpoNmutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only themptACDoperon was shown to be directly controlled byσ54. Therefore, the remaining modifications seem to be due to indirect effects. In parallel, anin silicoanalysis suggests thatσ54may directly control the expression of four different phosphotransferase system (PTS) operons, includingmptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest thatσ54is mainly involved in the control of carbohydrate metabolism inL. monocytogenesvia direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation.
Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export
Authors: Wyborn Neil R. , Clark Angela , Roberts Ruth E. , Jamieson Stuart J. , Tzokov Svetomir , Bullough Per A. , Stillman Timothy J. , Artymiuk Peter J. , Galen James E. , Zhao Licheng , Levine Myron M. , Green Jeffrey
Published: May 2004

Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic β-hairpin structure (the β-tongue, residues 177–203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the β-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36–41 Å diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.
Inability of Pneumocystis organisms to incorporate bromodeoxyuridine suggests the absence of a salvage pathway for thymidine
Authors: Vestereng Vibeke H. , Kovacs Joseph A.
Published: May 2004

Because thymidine metabolism is a potential target for therapy ofPneumocystispneumonia, it was investigated whetherPneumocystisorganisms have a salvage pathway for thymidine by administering 5-bromo-2′-deoxyuridine (BrdU) to mice and rats withPneumocystispneumonia. Although BrdU incorporation was detected in host cells, no incorporation was seen inPneumocystisorganisms infecting either rats or mice. This suggests thatPneumocystisorganisms do not have a salvage pathway for thymidine, and that inhibitors ofde novosynthesis, such as thymidylate synthase inhibitors, may be effective drugs for treatingPneumocystispneumonia.
Differential expression and interaction of transcription co-activator MBF1 with TATA-binding protein (TBP) in the apicomplexan Cryptosporidium parvum
Authors: Millership Jason J. , Waghela Palvi , Cai Xiaomin , Cockerham Amy , Zhu Guan
Published: May 2004

All gene-specific transcriptional activators initiate gene transcriptions by binding to promoter sequences and recruiting general transcription factors including TATA-binding protein (TBP) to upstream of targeted genes. Some of them require multiprotein bridging factors (MBFs); for example, the type 1 MBF (MBF1) which interconnects the gene activator with TBP. In this study, the properties of a previously cloned type 1 multiprotein bridging factor (CpMBF1) and a newly identified TBP (CpTBP1) from the apicomplexanCryptosporidium parvumwere investigated. Genes encoding both proteins were differentially expressed as determined by semi-quantitative RT-PCRs during the parasite life cycle, but in different patterns. The highest level of expression ofCpMBF1was in the well-developed intracellular parasites, whereas that ofCpTBP1was found in intact oocysts and late intracellular stages, possibly correlated with the formation of oocysts. Both CpMBF1 and CpTBP1 were expressed as maltose-binding protein fusion proteins. The function of CpTBP1 was confirmed by its ability to bind a biotinylated DNA oligonucleotide containing TATA consensus sequence. The interaction between CpMBF1 and CpTBP1 was also observed by an electrophoretic mobility shift assay. Since little is known about the regulation and control of gene activity inC. parvum, this study may point to a new direction for the study of gene activation associated with the development of the complex life cycle of this parasite.
Intron-containing β-tubulin transcripts in Cryptosporidium parvum cultured in vitro
Authors: Cai Xiaomin , Lancto Cheryl A. , Abrahamsen Mitchell S. , Zhu Guan
Published: May 2004

The genome ofCryptosporidium parvumcontains a relatively small number of introns, which includes theβ-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of theβ-tubulin transcripts in the late life cycle stages culturedin vitro. Although normally splicedβ-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected withC. parvumfor 12–72 h), at 48–72 h post-infection unprocessedβ-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescencein situhybridization (FISH) using an intron-specific probe. The intron-containingβ-tubulin transcripts appeared unique to thein vitro-culturedC. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages ofC. parvumare partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest ofC. parvumdevelopmentin vitro. This unusual observation prompts speculation that the abnormal intron-splicing ofβ-tubulin transcripts may be one of the factors preventing complete development of this parasitein vitro. Furthermore, the presence of both processed and unprocessed introns inβ-tubulin transcriptsin vitromay provide a venue for studying overall mechanisms for intron-splicing in this parasite.
Similar genotypes of Pneumocystis jirovecii in different forms of Pneumocystis infection
Authors: Totet Anne , Duwat Hyacinthe , Magois Eline , Jounieaux Vincent , Roux Patricia , Raccurt Christian , Nevez Gilles
Published: May 2004

This study describes the genotyping of Pneumocystis jirovecii organisms isolated from three groups of patients that developed diverse forms of P. jirovecii infection; the patients were monitored in the same French hospital. Forty archival specimens from 13 adults with Pneumocystis pneumonia, eight adults colonized by P. jirovecii and 19 immunocompetent infants infected with the fungus contemporaneously with a bronchiolitis episode were analysed retrospectively. Genotyping was performed by analysis of sequences of the internal transcribed spacer (ITS)1 and ITS2 regions, and of the dihydropteroate synthase (DHPS) locus. At the ITS regions, a high diversity of genotypes, identical main genotypes (B1a3 and B2a1) and the occurrence of mixed infections (more than one genotype) were observed in the three patient groups. At the DHPS locus, the results indicated the presence of mutants in the two adult groups, as well as in the infant group. Consequently, at these two independent genomic regions, P. jirovecii isolates from patients who developed different forms of infection and who lived in the same geographical region presented common characteristics. These results suggest that patients infected with P. jirovecii, whatever the form of infection they present, are part of a common human reservoir for P. jirovecii.
Phylogenetic identification of Pneumocystis murina sp. nov., a new species in laboratory mice
Authors: Keely Scott P. , Fischer Jared M. , Cushion Melanie T. , Stringer James R.
Published: May 2004

Pneumocystisis a fungal genus that contains multiple species. One member of the genus that has not been formally analysed for its phylogenetic relationships and possible species status is thePneumocystisfound in laboratory mice,Pneumocystis murinasp. nov. (type strain ATCC PRA-111T=CBS 114898T), formerly known asPneumocystis cariniif. sp.muris. To advance research in this area, approximately 3000 bp of additional DNA sequence were obtained from the locus encoding rRNAs. This sequence and others were used to determine genetic distances betweenP. murinaand other members of the genus. These distances indicated thatP. murinaDNA is most similar to that of the species ofPneumocystisfound in laboratory rats. Nevertheless,P. murinais at least as diverged from these otherPneumocystisspecies as species in other fungal genera are from each other. The 18S rRNA gene sequence divergence exhibited byP. murinacould not be ascribed to accelerated evolution of this gene as similar levels of divergence were observed at seven other loci. When five genes were used to construct phylogenetic trees for fivePneumocystistaxa, includingP. murina, all the trees had the same topology, indicating that genes do not flow among these taxa. The gene trees were all strongly supported by statistical tests. When sequences from the rRNA-encoding locus were used to estimate the time of divergence ofP. murina, the results indicated thatP. murinais as old as the mouse. Taken together, these data support previous recognition of multiple species in the genus and indicate thatP. murinais a phylogenetic species as well.
Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall
Authors: Karr Craig D. , Jarroll Edward L.
Published: May 2004

Uridine-5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall ofGiardiaand is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encystingGiardiais reported, and this activity exclusively incorporates [1-14C]GalNAc (from UDP-[14C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall ofGiardia. This ethanol precipitate exhibits characteristics ofGiardiacyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose,d-glucosamine andd-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparentKmof 0·048 mM for UDP-GalNAc, aVmaxof 0·70 nmol min−1(mg protein)−1and a requirement for divalent cations in the following order of preference: Ca2+, Mg2+>Co2+>>>Mn2+, Zn2+. EDTA inhibits CWS activity.
Mitosomes of Entamoeba histolytica are abundant mitochondrion-related remnant organelles that lack a detectable organellar genome
Authors: León-Avila Gloria , Tovar Jorge
Published: May 2004

The existence of mitochondrion-related relict organelles (mitosomes) in the amitochondrial human pathogenEntamoeba histolyticaand the detection of extranuclear DNA-containing cytoplasmic structures (EhKOs) has led to the suggestion that a remnant genome from the original mitochondrial endosymbiont might have been retained in this organism. This study reports on the mutually exclusive distribution of Cpn60 and extranuclear DNA inE. histolyticaand on the distribution of Cpn60-containing mitosomes in this parasite.In situnick-translation coupled to immunofluorescence microscopy failed to detect the presence of DNA in mitosomes, either in fixed parasite trophozoites or in partially purified organellar fractions. These results indicate that a remnant organellar genome has not been retained inE. histolyticamitosomes and demonstrate unequivocally that EhKOs and mitosomes are distinct and unrelated cellular structures.
Cryptococcus neoformans Ilv2p confers resistance to sulfometuron methyl and is required for survival at 37 °C and in vivo
Authors: Kingsbury Joanne M. , Yang Zhonghui , Ganous Tonya M. , Cox Gary M. , McCusker John H.
Published: May 2004

Acetolactate synthase catalyses the first common step in isoleucine and valine biosynthesis and is the target of several classes of inhibitors. TheCryptococcus neoformans ILV2gene, encoding acetolactate synthase, was identified by complementation of aSaccharomyces cerevisiae ilv2mutant.C. neoformansis highly resistant to the commercially available acetolactate synthase inhibitor, sulfometuron methyl (SM). Expression ofC. neoformans ILV2inS. cerevisiaeconferred SM resistance, indicating that the SM resistance ofC. neoformansis due, at least in part, toC. neoformansIlv2p. TheC. neoformans ILV2gene was disrupted. Theilv2mutants were auxotrophic for isoleucine and valine and the auxotrophy was satisfied by these amino acids only when proline, and not ammonium, was the nitrogen source, indicating nitrogen regulation of amino acid transport.ilv2mutants rapidly lost viability at 37 °C and when starved for isoleucine and valine. Consistent with these phenotypes, anilv2mutant was avirulent and unable to survive in mice. BecauseC. neoformansIlv2p is required for virulence and survivalin vivo, inhibitors of branched-chain amino acid biosynthesis may make valuable antifungal agents.
Expression in fibroblasts and in live animals of Entamoeba histolytica polypeptides EhCP112 and EhADH112
Authors: Madriz Xochil , Martínez Máximo B. , Rodríguez Mario A. , Sierra Gustavo , Martínez-López Carolina , Riverón Ana M. , Flores Leopoldo , Orozco Esther
Published: May 2004

EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved inEntamoeba histolytica's cytopathic effect, target-cell adherence and phagocytosis. The EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles. Here, the independent expression of EhCP112 and EhADH112 in fibroblasts and hamsters was analysed. Also investigated was the immunological response in animals independently inoculated with plasmid pcDNA-Ehcp112, which carries the complete cysteine protease-encoding gene, or with plasmid pcDNA-Ehadh112, which carries the C terminus of the adhesin-encoding gene, or with a mixture of both. Both proteins were expressed in the plasma membranes of the transfected fibroblasts. EhCP112 was toxic for the mammalian cells. Proteins were also independently expressed in hamsters after inoculation with the plasmids. Their expression was indirectly evaluated by the presence of antibodies in the inoculated animals. Remarkably, co-immunization of the animals with the two DNA plasmids resulted in an earlier and higher anti-E. histolyticaIgG induction than immunization with separate plasmids. In contrast, the cellular immune response was not noticeably improved by the plasmid mixture. Interestingly, protection against liver abscesses was detected only in animals that received the plasmid mixture and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112or pcDNA-Ehadh112.
Honda’s Global Flexifactory Network
Authors: Mair Andrew
Published: May 2004

Flexifactories, where the product made can be changed at low cost and
quickly, constitute an important aspect of manufacturing flexibility.
Examination of Honda flexifactories worldwide reveals how models are
changed over, the model mix altered, and whole factories switched to
making new types of products. Flexifactories are logically operated as
networks, constituting a bridge between the “micro” flexibility of people
and machines and overall corporate flexibility, resulting in improved
mass production. This is not to argue that the flexibility of
flexifactories is without limit, nor that they do not contain their own
rigidities. Further empirical and theoretical research is needed to
compare the flexifactory model with previous models of optimal factory
organization.
Total Effectiveness in a Just‐in‐Time System
Authors: Karlsson Christer , Norr Christer
Published: May 2004

Discusses the effects of the implementation of Just‐in‐Time (JIT) in the
relationships between suppliers and manufacturing companies within
Swedish industry. Explores whether such relationships will be beneficial
for all parties and create higher total economic effectiveness in the
supply relation or if the often more powerful buyer only transfers its
stock holding problems to the smaller and weaker part in the
buyer‐supplier relation. Two buyer‐supplier relations in the automobile
industry are studied, one more from the buyer′s point of view and the
other from the supplier′s side. Proves that there are very clear
possibilities to get higher total effectiveness in such relations and
that mutual benefits are really obtainable. Shows the increased
importance of reduced arrival variancy but also that JIT does not
necessarily mean an increased traffic intensity. Introduces an important
finding by identifying the time interval between the Sequence Locking
Instant and the point when the component is needed in Production (SLIP)
as the determining factor for JIT relations rather than the physical
distance per se.
Realizing Strategy through Measurement
Authors: Neely Andy , Mills John , Platts Ken , Gregory Mike , Richards Huw
Published: May 2004

It is widely accepted that firms should have manufacturing strategies
consistent with their overall business strategies, but while much has
been written about the content of these strategies, little has been said
about how they should be developed and realized. Reports on research
which set out to investigate the extent to which UK firms seek to
influence the realization of their manufacturing strategies through
their performance measurement systems. Over 800 small and medium‐sized
manufacturing enterprises (SMEs) in the UK were surveyed in late 1992.
Analysis of the data suggests that while firms which compete on quality
or time place most emphasis on performance measures which match their
strategies, those which compete on price do not.
Frameworks for Understanding Business Process Re‐engineering
Authors: Childe S.J. , Maull R.S. , Bennett J.
Published: May 2004

Introduces a number of concepts and frameworks developed during the
first phase of an EPSRC/CDP grant entitled “A Specification of a Business
Process Re‐engineering Methodology for Small and Medium Sized
Manufacturing Enterprises”. A traditional, functionally oriented
organization is characterized by fragmentation and staged inventories of
work caused by a failure to integrate and synchronize activities. A
process focused organization aims to optimize performance across the
whole process. Outlines many of the key issues facing organizations
seeking to adopt a process orientation. Presents two frameworks for
analysis. The first identifies the major business processes in any
manufacturing company, the second seeks to classify types of business
process re‐engineering intervention. Concludes by discussing the major
issues currently facing those researching into business process
re‐engineering.
Managers’ perceptions of the importance of supply, overhead and
operating costs
Authors: Armistead Colin , Bowman Cliff , Newton Julia
Published: May 2004

Examines the way in which managers perceive the relative importance
of the three costs as applied to professional service firms, financial
services firms and retail distribution and manufacturing. Managers′
perceptions may be determined by the nature of the business, the
operational focus and the opportunities for the control of costs.
Examines the existing literature for indications of the importance
attached to each of these costs. Concludes that managers have more
discretion of the control of operating costs where they represent a high
proportion of the three types and where supply and overhead costs form a
high proportion of total costs, managers can exercise discretion by
challenging existing industry recipes through restructuring their
operations.
A planning model for just‐in‐time batch manufacturing
Authors: Houghton Erne , Portougal Victor
Published: May 2004

Addresses the problem of implementing just‐in‐time (JIT) principles
in a processing shop organized in a batch manufacturing environment. The
focus is on a processing shop separated from an assembly shop by a parts
store. Presents a multi‐criteria variant of the capacitated lot‐sizing
model. The approach involves a pre‐emptive priority for JIT processing
schedules, and a secondary priority for desirable load profiles.
Solution properties are derived and used to facilitate the solution
process for both the primary and secondary criterion models. Load
profiles are assessed in the context of capacity requirements planning
as well as from the alternative viewpoint of providing feedback to
capacity planning through a consideration of capacity cushions. At both
planning levels the approach is shown to allow optimum resource
utilization without violating JIT principles. Presents optimization
procedures, including a heuristic with an analytic performance bound.
A mathematical programming model for a pull type ordering system
including lot production processes
Authors: Watanabe Norio , Hiraki Shusaku
Published: May 2004

Considers a multi‐stage multi‐product production, inventory and
transportation system including lot production processes and develops a
mathematical model for a pull type ordering system. The decision
variables of the presented model are initial ordering quantities and the
objective is to minimize the sum of the replenishment level at each
inventory point. The model is formulated as an integer programming
problem, and an approximate procedure is proposed to obtain a near
optimal solution in short time using a mathematical programming package.
Finally, shows a numerical example of the model applied to an actual
manufacturing system of an automobile parts manufacturer in order to
verify the effectiveness of the solution procedure and to clarify the
applicability of the modelling approach.
The optimal number of kanbans in a manufacturing system with
general machine breakdowns and stochastic demands
Authors: Yan Houmin
Published: May 2004

Considers manufacturing systems with a failure‐prone machine and a
stochastic demand. The production is controlled by a kanban
scheme. Although the kanban‐control enjoys many applications, in
a stochastic setting, the problem of defining the optimal number of
circulating kanbans remains unsolved except for some special
cases. Seeks an optimal kanban‐controlled policy which minimizes
long run average inventory and backlog costs. Uses perturbation analysis
to estimate the gradients of the cost functional with respect to the
number of circulating kanbans, and then employs an iterative
algorithm, which is a constant step‐size stochastic approximation
procedure, to find the optimal number of circulating kanbans.
Proves that the perturbed path is a shifted version of the nominal path,
and the gradient estimate is consistent. Also conducts numerical
experiments to investigate the performance of the proposed algorithms.
Comparative analysis of group scheduling heuristics in a flow shop
cellular system
Authors: Leu Bor‐Yuh , Nazemetz John W.
Published: May 2004

Investigates group scheduling heuristics in a flow shop cellular
system with two types of order shipment environment: permitted early
shipment (PES) and forbidden early shipment (FES). Once an order is
completed in the PES environment, it leaves the system without delay. In
the FES environment, however, the suppliers must deliver materials to
the system on a just‐in‐time basis; and a completed order cannot leave
the system earlier than the customer has specified. The FES environment
therefore captures many of the features of the currently popular
just‐in‐time (JIT) approach to manufacturing. Examines shop factors such
as the order shipment environment, demand pattern variability, and order
release policy. Shows that the performance of heuristics in the FES
environment differs from their performance in the PES environment,
except for tardiness‐related measures. Identifies the best performing
heuristics, which can provide guidance for schedulers or decision makers
in the selection of preferable heuristics.
Appraisal of JIT using financial measures
Authors: Vinodrai Pandya K. , Boyd J.
Published: May 2004

In the last 30 years many Japanese companies have set up plants in
the UK. This has brought many benefits, one of which being the
introduction of their unique style of management. The last decade has
seen an increasing use of these practices in the UK. A survey of the
Japanese companies based in the UK was carried out by a team of
researchers at the University of Strathclyde. Discusses the use of JIT,
one of the strategies examined in the survey.
Divestments in Public Enterprises: The Indian Experience
Authors: Sankar T.L. , Mishra R.K. , Lateef Syed Mohammed A.
Published: May 2004

Examines one of the most important reforms relating to public
enterprise (PE) policy in India, namely divestment of their
share‐holdings. Discusses the philosophy, process, organizational
mechanism, expectations and outcomes of divestment in PEs. Finally,
points out the major weaknesses retarding the success of the newly
introduced divestment policy and outlines some reformatory measures to
overcome them. As a backdrop, presents the historical background,
current scenario, and problems and performance of PEs in India, but has
been restricted to the central PEs, i.e. enterprises owned and managed
by the central government only.
The future of public sector management
Authors: Flynn Norman
Published: May 2004

If we try to look at the future of public sector management in the
UK, it might be more useful to think about what is happening in the rest
of Europe, rather than looking at the USA (“reinventing
government”, etc.) and especially New Zealand. There are some
common pressures on the public sector in Europe, but not a unified or
convergent approach to management. In the areas of structural
arrangements about policy making and service delivery, relationships
between tiers of government, goal setting, planning, budgeting,
evaluation, “user” orientation and personnel management,
there are choices to be made.