bluuebunny/crossref_metadata_2025 huggingface dataset:
End‐damage‐specific proteins facilitate recruitment or stability of X‐ray cross‐complementing protein 1 at the sites of DNA single‐strand break repair
Authors: Parsons Jason L. , Dianova Irina I. , Boswell Emma , Weinfeld Michael , Dianov Grigory L.
Published: November 2005

Ionizing radiation, oxidative stress and endogenous DNA‐damage processing can result in a variety of single‐strand breaks with modified 5′ and/or 3′ ends. These are thought to be one of the most persistent forms of DNA damage and may threaten cell survival. This study addresses the mechanism involved in recognition and processing of DNA strand breaks containing modified 3′ ends. Using a DNA–protein cross‐linking assay, we followed the proteins involved in the repair of oligonucleotide duplexes containing strand breaks with a phosphate or phosphoglycolate group at the 3′ end. We found that, in human whole cell extracts, end‐damage‐specific proteins (apurinic/apyrimidinic endonuclease 1 and polynucleotide kinase in the case of 3′ ends containing phosphoglycolate and phosphate, respectively) which recognize and process 3′‐end‐modified DNA strand breaks are required for efficient recruitment of X‐ray cross‐complementing protein 1–DNA ligase IIIα heterodimer to the sites of DNA repair.
Molecular analysis of the interaction between cardosin A and phospholipase Dα
Authors: Simões Isaura , Mueller Eva‐Christina , Otto Albrecht , Bur Daniel , Cheung Alice Y. , Faro Carlos , Pires Euclides
Published: November 2005

Cardosin A is an RGD‐containing aspartic proteinase from the stigmatic papillae of Cynara cardunculus L. A putative cardosin A‐binding protein has previously been isolated from pollen suggesting its potential involvement in pollen–pistil interaction [Faro C, Ramalho‐Santos M, Vieira M, Mendes A, Simões I, Andrade R, Verissimo P, Lin X, Tang J & Pires E (1999) J Biol Chem274, 28724–28729]. Here we report the identification of phospholipase Dα as a cardosin A‐binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull‐down assays. To investigate the structural and molecular determinants involved in the interaction, pull‐down assays with cardosin A and various glutathione S‐transferase‐fused phospholipase Dα constructs were performed. Results revealed that the C2 domain of phospholipase Dα contains the cardosin A‐binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge‐wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dα can act as a cardosin A‐binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE‐recognition domains.
From heart to mind
Authors: Nothacker Hans‐Peter , Clark Stewart
Published: November 2005

The discovery of novel biologically active peptides has led to an explosion in our understanding of the molecular mechanisms that underlie the regulation of sleep and wakefulness. Urotensin II (UII), a peptide originally isolated from fish and known for its strong cardiovascular effects in mammals, is another surprising candidate in the regulatory network of sleep. The UII receptor was found to be expressed by cholinergic neurons of laterodorsal and pedunculopontine tegmental nuclei, an area known to be of utmost importance for the on‐ and offset of rapid eye movement (REM) sleep. Recently, physiological data have provided further evidence that UII is indeed a modulator of REM sleep. The peptide directly excites cholinergic mesopontine neurons and increases the rate of REM sleep episodes. These new results and its emerging behavioral effects establish UII as a neurotransmitter/neuromodulator in mammals and should spark further interest into the neurobiological role of the peptide.
Characterization of the interaction between the plasma membrane H+‐ATPase of Arabidopsis thaliana and a novel interactor (PPI1)
Authors: Viotti Corrado , Luoni Laura , Morandini Piero , De Michelis Maria Ida
Published: November 2005

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C‐terminus of Arabidopsis thaliana plasma membrane H+‐ATPase (EC 3.6.3.6) (Morandini P, Valera M, Albumi C, Bonza MC, Giacometti S, Ravera G, Murgia I, Soave C & De Michelis MI (2002) Plant J31, 487–497). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H+‐ATPase. PPI1‐induced stimulation of H+‐ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH‐independent when the enzyme C‐terminus was displaced by fusicoccin‐induced binding of 14‐3‐3 proteins. The latter treatment did not affect PPI1 affinity for the H+‐ATPase. These results indicate that PPI1 can bind the H+‐ATPase independently of the C‐terminus conformation, but is not able to suppress the C‐terminus auto‐inhibitory action.
Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis
Authors: Hothi Parvinder , Khadra Khalid Abu , Combe Jonathan P. , Leys David , Scrutton Nigel S.
Published: November 2005

The heterologous expression of tryptophan trytophylquinone (TTQ)‐dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf‐2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad‐host‐range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady‐state reaction profiles for recombinant AADH as a function of substrate concentration differed between ‘fast’ (tryptamine) and ‘slow’ (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature‐dependent kinetic isotope effect indicated that C‐H/C‐D bond breakage is only partially rate‐limiting in steady‐state reactions with benzylamine. Stopped‐flow studies of the reductive half‐reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady‐state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C‐H bond by quantum mechanical tunnelling. The limiting rate constant (klim) for TTQ reduction is controlled by a single ionization with pKa value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of klim/Kd of pKa values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential ionizations is discussed.
Interpreting Velocities from Heat‐Based Flow Sensors by Numerical Simulation
Authors: Su Grace W. , Freifeld Barry M. , Oldenburg Curtis M. , Jordan Preston D. , Daley Paul F.
Published: November 2005

We have carried out numerical simulations of three‐dimensional nonisothermal flow around an in situ heat‐based flow sensor to investigate how formation heterogeneities can affect the interpretation of ground water flow velocities from this instrument. The flow sensor operates by constant heating of a 0.75‐m‐long, 5‐cm‐diameter cylindrical probe, which contains 30 thermistors in contact with the formation. The temperature evolution at each thermistor can be inverted to obtain an estimate of the ground water flow velocity vector using the standard interpretive method, which assumes that the formation is homogeneous. Analysis of data from heat‐based flow sensors installed in a sand aquifer at the Former Fort Ord Army Base near Monterey, California, suggested an unexpected component of downward flow. The magnitudes of the vertical velocities were expected to be much less than those of the horizontal velocities at this site because the sensors were installed just above a clay aquitard. Numerical simulations were conducted to examine how differences in thermal conductivities may lead to spurious indications of vertical flow velocities. We found that a decrease in the thermal conductivity near the bottom of the sensor can perturb the temperature profiles along the instrument in such a manner that analyses assuming homogeneous thermal conductivity could indicate a vertical flow component even though flow is actually horizontal. This work demonstrates how modeling can be used to simulate instrument response to formation heterogeneity and shows that caution must be used in interpreting data from such devices.
Cyclic AMP-Independent Regulation of Protein Kinase A Substrate Phosphorylation by Kelch Repeat Proteins
Authors: Lu Ailan , Hirsch Jeanne P.
Published: November 2005

Pseudohyphal and invasive growth in the yeast
Saccharomyces cerevisiae
is regulated by the kelch repeat-containing proteins Gpb1p and Gpb2p, which act downstream of the G protein α-subunit Gpa2p. Here we show that deletion of
GPB1
and
GPB2
causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing
gpb1
Δ
gpb2
Δ mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by
gpb1
Δ
gpb2
Δ mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.
Gpi19, the
Saccharomyces cerevisiae
Homologue of Mammalian PIG-P, Is a Subunit of the Initial Enzyme for Glycosylphosphatidylinositol Anchor Biosynthesis
Authors: Newman Heather A. , Romeo Martin J. , Lewis Sarah E. , Yan Benjamin C. , Orlean Peter , Levin David E.
Published: November 2005

Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of
N
-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-
N
-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast
Saccharomyces cerevisiae
. The uncharacterized yeast gene
YDR437w
encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene
GPI19
. Similar to other mutants in the yeast enzyme, temperature-sensitive
gpi19
mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional
gpi19
mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.
Group III Histidine Kinase Is a Positive Regulator of Hog1-Type Mitogen-Activated Protein Kinase in Filamentous Fungi
Authors: Yoshimi Akira , Kojima Kaihei , Takano Yoshitaka , Tanaka Chihiro
Published: November 2005

We previously reported that the group III histidine kinase Dic1p in the maize pathogen
Cochliobolus heterostrophus
is involved in resistance to dicarboximide and phenylpyrrole fungicides and in osmotic adaptation. In addition, exposure to the phenylpyrrole fungicide fludioxonil led to improper activation of Hog1-type mitogen-activated protein kinases (MAPKs) in some phytopathogenic fungi, including
C. heterostrophus
. Here we report, for the first time, the relationship between the group III histidine kinase and Hog1-related MAPK: group III histidine kinase is a positive regulator of Hog1-related MAPK in filamentous fungi. The phosphorylation pattern of
C. heterostrophus
BmHog1p (Hog1-type MAPK) was analyzed in wild-type and
dic1-
deficient strains by Western blotting. In the wild-type strain, phosphorylated BmHog1p was detected after exposure to both iprodione and fludioxonil at a concentration of 1 μg/ml. In the
dic1-
deficient strains, phosphorylated BmHog1p was not detected after exposure to 10 μg/ml of the fungicides. In response to osmotic stress (0.4 M KCl), a trace of phosphorylated BmHog1p was found in the
dic1-
deficient strains, whereas the band representing active BmHog1p was clearly detected in the wild-type strain. Similar results were obtained for
Neurospora crassa
Os-2p MAPK phosphorylation in the mutant of the group III histidine kinase gene
os-1
. These results indicate that group III histidine kinase positively regulates the activation of Hog1-type MAPKs in filamentous fungi. Notably, the Hog1-type MAPKs were activated at high fungicide (100 μg/ml) and osmotic stress (0.8 M KCl) levels in the histidine kinase mutants of both fungi, suggesting that another signaling pathway activates Hog1-type MAPKs in these conditions.
Saccharomyces cerevisiae
Npc2p Is a Functionally Conserved Homologue of the Human Niemann-Pick Disease Type C 2 Protein, hNPC2
Authors: Berger Adam C. , Vanderford Thomas H. , Gernert Kim M. , Nichols J. Wylie , Faundez Victor , Corbett Anita H.
Published: November 2005

Niemann-Pick Disease Type C (NP-C) is a fatal neurodegenerative disease, which is biochemically distinguished by the lysosomal accumulation of exogenously derived cholesterol. Mutation of either the
hNPC1
or
hNPC2
gene is causative for NP-C. We report the identification of the yeast homologue of human NPC2,
Saccharomyces cerevisiae
Npc2p. We demonstrate that scNpc2p is evolutionarily related to the mammalian NPC2 family of proteins. We also show, through colocalization, subcellular fractionation, and secretion analyses, that yeast Npc2p is treated similarly to human NPC2 when expressed in mammalian cells. Importantly, we show that yeast Npc2p can efficiently revert the unesterified cholesterol and GM1 accumulation seen in
hNPC2

−/−

patient fibroblasts demonstrating that it is a functional homologue of human NPC2. The present study reveals that the fundamental process of NPC2-mediated lipid transport has been maintained throughout evolution.
Independent Metalloregulation of Ace1 and Mac1 in
Saccharomyces cerevisiae
Authors: Keller Greg , Bird Amanda , Winge Dennis R.
Published: November 2005

Ace1 and Mac1 undergo reciprocal copper metalloregulation in yeast cells. Mac1 is functional as a transcriptional activator in copper-deficient cells, whereas Ace1 is a transcriptional activator in copper-replete cells. Cells undergoing a transition from copper-deficient to copper-sufficient conditions through a switch in the growth medium show a rapid inactivation of Mac1 and a corresponding rise in Ace1 activation. Cells analyzed after the transition show a massive accumulation of cellular copper. Under these copper shock conditions we show, using two epitope-tagged variants of Mac1, that copper-mediated inhibition of Mac function is independent of induced protein turnover. The transcription activity of Mac1 is rapidly inhibited in the copper-replete cells, whereas chromatin immunoprecipitation studies showed only partial copper-induced loss of DNA binding. Thus, the initial event in copper inhibition of Mac1 function is likely copper inhibition of the transactivation activity. Copper inhibition of Mac1 in transition experiments is largely unaffected in cells overexpressing copper-binding proteins within the nucleus. Likewise, high expression of a copper-binding, non-DNA-binding Mac1 mutant is without effect on the copper activation of Ace1. Thus, metalloregulation of Ace1 and Mac1 occurs independently.
Repressors Nrg1 and Nrg2 Regulate a Set of Stress-Responsive Genes in
Saccharomyces cerevisiae
Authors: Vyas Valmik K. , Berkey Cristin D. , Miyao Takenori , Carlson Marian
Published: November 2005

The yeast
Saccharomyces cerevisiae
responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in
nrg1Δ nrg2Δ
double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of
NRG1
and
NRG2
enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contribute to repression of target genes in the absence of stress but also limit induction in response to salt stress. We suggest that Nrg1/Nrg2 fine-tune the regulation of a set of stress-responsive genes.
A Rho-Type GTPase,
rho-4
, Is Required for Septation in
Neurospora crassa
Authors: Rasmussen Carolyn G. , Glass N. Louise
Published: November 2005

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division, and reorganization of cytoskeletal elements. Phylogenetic analysis of predicted fungal Rho proteins suggests that a new Rho-type GTPase family, whose founding member is Rho4 from the archiascomycete
Schizosaccharomyces pombe
, is involved in septation.
S. pombe rho4
Δ mutants have multiple, abnormal septa. In contrast to
S. pombe rho4
Δ mutants, we show that strains containing
rho-4
loss-of-function mutations in the filamentous fungus
Neurospora crassa
lead to a loss of septation. Epitope-tagged RHO-4 localized to septa and to the plasma membrane. In other fungi, the steps required for septation include formin, septin, and actin localization followed by cell wall synthesis and the completion of septation.
rho-4
mutants were unable to form actin rings, showing that RHO-4 is required for actin ring formation. Characterization of strains containing activated alleles of
rho-4
showed that RHO-4-GTP is likely to initiate new septum formation in
N. crassa
.
Trichomonas vaginalis
Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells
Authors: Bastida-Corcuera Felix D. , Okumura Cheryl Y. , Colocoussi Angie , Johnson Patricia J.
Published: November 2005

The extracellular human pathogen
Trichomonas vaginalis
is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced
T. vaginalis
LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins
Ricinnus comunis
agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of
T. vaginalis
parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of
T. vaginalis
. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that
T. vaginalis
adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.
Could Human Papillomaviruses Be Spread through Blood?
Authors: Bodaghi Sohrab , Wood Lauren V. , Roby Gregg , Ryder Celia , Steinberg Seth M. , Zheng Zhi-Ming
Published: November 2005

The
human papillomaviruses (HPVs) are epitheliotropic viruses that require
the environment of a differentiating squamous epithelium for their life
cycle. HPV infection through abrasion of the skin or sexual intercourse
causes benign warts and sometimes cancer. HPV DNA detected in the blood
has been interpreted as having originated from metastasized cancer
cells. The present study examined HPV DNA in banked, frozen peripheral
blood mononuclear cells (PBMCs) from 57 U.S. human immunodeficiency
virus (HIV)-infected pediatric patients collected between 1987 and 1996
and in fresh PBMCs from 19 healthy blood donors collected in 2002 to
2003. Eight patients and three blood donors were positive mostly for
two subgroups of the HPV type 16 genome. The HPV genome detected in all
11 PBMC samples existed as an episomal form, albeit at a low DNA copy
number. Among the eight patients, seven acquired HIV from transfusion
(three associated with hemophilia) and one acquired HIV through
vertical transmission; this patient also had received a transfusion
before sampling. Our data suggest that PBMCs may be HPV carriers and
might spread the virus through
blood.
Development of One-Step, Real-Time, Quantitative Reverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E
Authors: Vijgen Leen , Keyaerts Els , Moës Elien , Maes Piet , Duson Griet , Van Ranst Marc
Published: November 2005

The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a routine clinical setting. In this study, we present a very sensitive and specific TaqMan-based, real-time quantitative reverse transcriptase PCR (qRT-PCR) for the rapid detection and quantitation of human coronaviruses (HCoVs) OC43 and 229E. Absolute viral load measurement in clinical samples was achieved through the construction of in-house HCoV OC43 and 229E cRNA standards for the generation of a standard curve. The HCoV OC43 assay allows quantitation over a range from 20 to 2 × 10
8
RNA copies per reaction mixture (5 μl RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 10
3
to 10
10
viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 × 10
9
per reaction mixture can be detected, which corresponds to 10
4
to 10
11
viral genome equivalents per ml sample. A total of 100 respiratory samples screened for the presence of HCoVs OC43 and 229E by using conventional RT-PCR were assessed in parallel by the qRT-PCR assays. By use of the real-time qRT-PCR techniques, the detection rate of HCoVs OC43 and 229E increased from 2.0% to 3.1% and from 0.3% to 2.5%, respectively. The real-time qRT-PCR assays described here allow the rapid, specific, and sensitive laboratory detection and quantitation of human coronaviruses OC43 and 229E.
Assessment of Band-Based Similarity Coefficients for Automatic Type and Subtype Classification of Microbial Isolates Analyzed by Pulsed-Field Gel Electrophoresis
Authors: Carriço J. A. , Pinto F. R. , Simas C. , Nunes S. , Sousa N. G. , Frazão N. , de Lencastre H. , Almeida J. S.
Published: November 2005

Pulsed-field gel electrophoresis (PFGE) has been the typing method of choice for strain identification in epidemiological studies of several bacterial species of medical importance. The usual procedure for the comparison of strains and assignment of strain type and subtype relies on visual assessment of band difference number, followed by an incremental assignment to the group hosting the most similar type previously seen. Band-based similarity coefficients, such as the Dice or the Jaccard coefficient, are then used for dendrogram construction, which provides a quantitative assessment of strain similarity. PFGE type assignment is based on the definition of a threshold linkage value, below which strains are assigned to the same group. This is typically performed empirically by inspecting the hierarchical cluster analysis dendrogram containing the strains of interest. This approach has the problem that the threshold value selected is dependent on the linkage method used for dendrogram construction. Furthermore, the use of a linkage method skews the original similarity values between strains. In this paper we assess the goodness of classification of several band-based similarity coefficients by comparing it with the band difference number for PFGE type and subtype classification using receiver operating characteristic curves. The procedure described was applied to a collection of PFGE results for 1,798 isolates of
Streptococcus pneumoniae
, which documented 96 types and 396 subtypes. The band-based similarity coefficients were found to perform equally well for type classification, but with different proportions of false-positive and false-negative classifications in their minimal false discovery rate when they were used for subtype classification.
Use of Full-Length Recombinant Calflagin and Its C Fragment for Improvement of Diagnosis ofTrypanosoma cruziInfection
Authors: Marcipar Iván S. , Roodveldt Cintia , Corradi Gerardo , Cabeza María L. , Brito Maria Edileuza F. , Winter Lucile M. Floeter , Marcipar Alberto J. , Silber Ariel M.
Published: November 2005

Serological diagnosis ofTrypanosoma cruziinfection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such asLeishmaniaspp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection byTrypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins inLeishmania majorwith the N-terminal moieties presenting high similarity withT. cruzigenes. This fact raised questions about their role in some cross-recognition of this antigen by sera fromLeishmaniaspp.-infected individuals. The completeT. cruzicalflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples fromT. cruzi, andLeishmania-infected patients. We were able to show that sera fromLeishmania(Viannia)braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera fromT. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected byT. cruzi, including 15 sera from people infected withL. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis ofT. cruziinfection in humans in geographic areas whereLeishmaniaspp. infection is coendemic.
Evaluation of Enzyme-Linked Immunosorbent Assays, an Immunofluorescent-Antibody Test, and Two Rapid Tests (Immunochromatographic-Dipstick and Gel Tests) for Serological Diagnosis of Symptomatic and Asymptomatic
Leishmania
Infections in Dogs
Authors: Mettler Maik , Grimm Felix , Capelli Gioia , Camp Heinrich , Deplazes Peter
Published: November 2005

Enzyme-linked immunosorbent assays (ELISAs) based on soluble antigens derived from promastigote or amastigote-like stages of
Leishmania infantum
and on the recombinant rK39 antigen, each in combination with different conjugates [anti-immunoglobulin G1 [IgG1], anti-IgG2, anti-IgG(γ), and anti-IgG heavy plus light chains], were compared to an immunofluorescent-antibody test (IFAT) and two commercially available rapid test systems (DiaMed-Vet-IT Leish and ID-PaGIA canine leishmaniasis antibody test) for the detection of specific anti-
Leishmania
antibodies in symptomatic and asymptomatic dogs with proven
L. infantum
infections. ELISAs based on soluble promastigote and amastigote antigens had very high sensitivities in symptomatic (
n
= 30; 100%) and asymptomatic dogs (
n
= 17; 94.1 to 100%), except when combined with the anti-IgG1 conjugate (41.2 to 82.4%). Specificities were high for all combinations (
n
= 50; 96 to 100%). The rK39 ELISA detected fewer asymptomatic cases (sensitivities, 52.9 to 64.7%) but was highly specific (96 to 100%). The IFAT was 90% sensitive in symptomatic dogs but was significantly less sensitive in asymptomatic cases (29.4%). However, it had an excellent specificity (100%). Test performances of the rapid tests based on the rK39 antigen were comparable to the ELISAs based on the same antigen. ELISAs based on soluble promastigote or amastigote antigens seem to be most suited for the serological diagnosis of canine
Leishmania
infections in both symptomatic and asymptomatic dogs. IFAT and the rK39 ELISA lack sensitivity in asymptomatic cases but are highly specific. Rapid tests like the rK39 dipstick test or the ID-PaGIA are helpful for confirming clinically suspected cases because of their high specificities in symptomatic animals.
Analysis of the Effect of DNA Purification on Detection of Human Papillomavirus in Oral Rinse Samples by PCR
Authors: D'Souza Gypsyamber , Sugar Elizabeth , Ruby William , Gravitt Patti , Gillison Maura
Published: November 2005

Human papillomavirus (HPV) has recently been associated with oral cancers. To prepare for a study of the natural history of oral HPV infection, the effect of the DNA purification method on HPV genomic DNA detection in Scope mouthwash oral rinse samples and the reproducibility of HPV detection in rinse samples collected 7 days apart were investigated. The study was conducted with a population at high risk for oral HPV infection: human immunodeficiency virus-infected men with CD4-cell counts <200. Five DNA purification methods were compared among equal aliquots of oral rinse samples collected from a subset of individuals. The purification methods included (i) proteinase K digestion (PKD) and heat inactivation; (ii) PKD and ethanol precipitation (EP); (iii) PKD, phenol-chloroform extraction, and EP; (iv) use of the Puregene DNA purification kit; and (v) use of the QIAamp DNA Blood Midi kit. HPV was detected by PCR amplification with PGMY09 and PGMY11 L1 primer pools and by use of a Roche linear array. Puregene-purified samples had higher human DNA yields and purities, and Puregene purification detected the greatest number of HPV-positive subjects and total HPV infections in comparison to the numbers detected by all other methods. The total number of HPV infections and HPV prevalence estimates were also higher for Puregene-processed oral rinse samples when a fixed volume (10 μl) rather than a fixed cell number (∼50,000 cells) was used for PCR amplification. A good concordance was observed for oral HPV infection status (agreement, 80%; kappa value, = 0.60) and type-specific infection (agreement, 98%; kappa value, 0.57) in matched oral rinse samples. The method of DNA purification significantly affects the detection of HPV genomic DNA from oral rinse samples and may result in exposure misclassification that could contribute to the inconsistent associations reported in the literature.
Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR
Authors: Beuselinck Kurt , van Ranst Marc , van Eldere J.
Published: November 2005

The performance of the
m
1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput “in-house” quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ± 0.06 for HCV and 0.97 ± 0.03 for HBV, indicating a linear extraction from 100 to 1.0 × 10
5
HCV IU/ml and from 100 to 1.0 × 10
6
HBV IU/ml. Intra- and interrun variability was below 0.23 log
10
IU/ml for 2.98 to 5.28 log
10
HCV IU/ml and 2.70 to 5.20 log
10
HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log
10
HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log
10
HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log
10
copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log
10
copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the
m
1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.
Development of a General-Primer-PCR-Reverse-Line-Blotting System for Detection of Beta and Gamma Cutaneous Human Papillomaviruses
Authors: Brink Antoinette A. T. P. , Lloveras Belen , Nindl Ingo , Heideman Daniëlle A. M. , Kramer Debbie , Pol René , Fuente M. Jose , Meijer Chris J. L. M. , Snijders Peter J. F.
Published: November 2005

The beta and gamma genera of papillomaviruses consist of epidermodysplasia verruciformis-related human papillomaviruses (HPVs) and phylogenetically related cutaneous HPVs. Here, we have developed a consensus primer PCR assay and reverse line blot typing system coupled thereto (referred to as beta and gamma cutaneous HPV PCR [BGC-PCR]) for detection and typing of 24 beta and gamma HPVs (HPV types 4, 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 48, 49, 50, 60, and 65). Because the HPV-specific PCR products are only 72 bp in size, the system is suitable for formalin-fixed, paraffin-embedded specimens and other samples in which the DNA is of suboptimal quality. This system was able to detect and type as little as 100 ag to 1 fg HPV DNA per reaction (depending on the HPV type) in a background of 100 ng human DNA without any cross-reactivity between the tested types. Beta and gamma HPVs were detected in DNA extracted from plucked eyebrow hairs of 31 of 34 renal transplant recipients. In addition, formalin-fixed, paraffin-embedded specimens from nonmelanoma skin tumors of renal transplant recipients (
n
= 25) and immunocompetent individuals (
n
= 15) scored BGC-PCR positive in 21 and 6 cases, respectively, with HPV type 5 (HPV5) and HPV8 being the predominant types. The data indicate that this method can be a valuable, user-friendly tool for the detection and typing of cutaneous HPV in clinical specimens and may have implications for future monitoring of vaccines or alternative treatment modalities for diseases caused by these cutaneous HPVs.
Population Structure and Properties of
Candida albicans
, as Determined by Multilocus Sequence Typing
Authors: Tavanti Arianna , Davidson Amanda D. , Fordyce Mark J. , Gow Neil A. R. , Maiden Martin C. J. , Odds Frank C.
Published: November 2005

We submitted a panel of 416 isolates of
Candida albicans
from separate sources to multilocus sequence typing (MLST). The data generated determined a population structure in which four major clades of closely related isolates were delineated, together with eight minor clades comprising five or more isolates. By Fisher's exact test, a statistically significant association was found between particular clades and the anatomical source, geographical source, ABC genotype, decade of isolation, and homozygosity versus heterozygosity at the mating type-like locus (MTL) of the isolates in the clade. However, these associations may have been influenced by confounding variables, since in a univariate analysis of variance, only the clade associations with ABC type and anatomical source emerged as statistically significant, providing the first indication of possible differences between
C. albicans
strain type clades and their propensity to infect or colonize different anatomical locations. There were no significant differences between clades with respect to distributions of isolates resistant to fluconazole, itraconazole, or flucytosine. However, the majority of flucytosine-resistant isolates belonged to clade 1, and these isolates, but not flucytosine-resistant isolates in other clades, bore a unique mutation in the
FUR1
gene that probably accounts for their resistance. A significantly higher proportion of isolates resistant to fluconazole, itraconazole, and flucytosine were homozygous at the MTL, suggesting that antifungal pressure may trigger a common mechanism that leads both to resistance and to MTL homozygosity. The utility of MLST for determining clade assignments of clinical isolates will form the basis for strain selection for future research into
C. albicans
virulence.
Fast Ligation-Mediated PCR, a Fast and Reliable Method for IS
6110
-Based Typing of
Mycobacterium tuberculosis
Complex
Authors: Reisig Florian , Kremer Kristin , Amthor Beate , van Soolingen Dick , Haas Walter H.
Published: November 2005

IS
6110
restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of
Mycobacterium tuberculosis
complex. We have previously described mixed-linker PCR, an IS
6110
-based PCR method that favorably compared with other typing methods for
M. tuberculosis
complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131
M. tuberculosis
complex and nontuberculous isolates showed the ability to differentiate 81 types among 90
M. tuberculosis
complex isolates with 84 different IS
6110
RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS
6110
RFLP. It may as well serve as a secondary typing technique for other, non-IS
6110
-based methods.
Characterization of Clonal Complexity in Tuberculosis by Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing
Authors: García de Viedma Darío , Alonso Rodriguez Noelia , Andrés Sandra , Ruiz Serrano Maria Jesús , Bouza Emilio
Published: November 2005

In recent years, the application of molecular tools has shown us that clonal complexity in infection byMycobacterium tuberculosisis not anecdotal. Exogenous reinfections, mixed infections, compartmentalization, and microevolution are different aspects of this issue. The detection and characterization of clonal variants ofM. tuberculosisby standard genotyping methods is laborious and frequently requires expertise. Our aim was to evaluate a new genotyping PCR-based method forM. tuberculosis, mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR), as a potential tool to simplify and optimize the clonal analysis of tuberculosis. MIRU-VNTR was able to detect mixed clonal variants in vitro, even for clones at low ratios (1:99). This technique was prospectively applied to search for cases infected by more than one clone. Clonal variants within the same host were detected in 3 out of 115 cases (2.6%), including cases with clones which were indistinguishable by restriction fragment length polymorphism or spoligotyping. In one case, coinfecting clonal variants differed in antibiotic susceptibilities. MIRU-VNTR was applied to cases with proven polyclonal infection, and it succeeded in detecting the coinfecting strains and proved useful in confirming cases of compartmentalized infection. MIRU-VNTR is a simple, rapid, and sensitive method which could facilitate and optimize the identification and characterization of clonal complexity inM. tuberculosisinfection.
Rapid and Accurate Genotyping of YMDD Motif Variants in the Hepatitis B Virus Genome by an Improved Reverse Dot Blot Method
Authors: Ou Zhi-Ying , Liu Na , Chen Chang-Jie , Cheng Gang , He Yun-Shao
Published: November 2005

By using the “flow-through hybridization” principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.
Human Infection with an Avian H9N2 Influenza A Virus in Hong Kong in 2003
Authors: Butt K. M. , Smith Gavin J. D. , Chen Honglin , Zhang L. J. , Leung Y. H. Connie , Xu K. M. , Lim Wilina , Webster Robert G. , Yuen K. Y. , Peiris J. S. Malik , Guan Yi
Published: November 2005

Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission.
Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of
Burkholderia pseudomallei
and
Burkholderia mallei
Authors: U'Ren Jana M. , Van Ert Matthew N. , Schupp James M. , Easterday W. Ryan , Simonson Tatum S. , Okinaka Richard T. , Pearson Talima , Keim Paul
Published: November 2005

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype
Burkholderia pseudomallei
and
Burkholderia mallei
isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as
Burkholderia thailandensis
and
Burkholderia cepacia
.
Distribution of Strain Families ofMycobacterium tuberculosisCausing Pulmonary and Extrapulmonary Disease in Hospitalized Children in Cape Town, South Africa
Authors: Nicol Mark P. , Sola Christophe , February Bradley , Rastogi Nalin , Steyn Lafras , Wilkinson Robert J.
Published: November 2005

We studied the association between strain family and extrapulmonary tuberculosis among 285 children presenting to a pediatric hospital. Extrapulmonary disease occurred in 56% of children without known human immunodeficiency virus infection, with meningitis accounting for 22% of the cases. Two strain families, LAM3/F11 and W-Beijing, predominated; but there was no overall association with extrapulmonary disease.
Phylogenetic Analysis of the p24-p7 Region of the Human Immunodeficiency Virus Type 1
gag
Gene To Determine Subtype Distribution among Female Sex Workers in Calcutta, India
Authors: Sengupta Satarupa , Jana Smarajit , Roy Pratim , Sarkar Kamalesh , Bhattacharya Sujit K. , Chakrabarti Sekhar
Published: November 2005

Human immunodeficiency virus type 1 (HIV-1) subtype C, based on the envelope region, has been reported to be predominant in India. We sequenced the p24-p7
gag
region from 51 HIV-1 seropositive female sex workers in Calcutta, India, for more-detailed molecular characterization. Subtype C was found to be prevalent, although no strong monophyletic cluster was observed.
Emergence of a
Streptococcus pneumoniae
Clinical Isolate Highly Resistant to Telithromycin and Fluoroquinolones
Authors: Faccone Diego , Andres Patricia , Galas Marcelo , Tokumoto Marta , Rosato Adriana , Corso Alejandra
Published: November 2005

Streptococcus pneumoniae
is a major pathogen causing community-acquired pneumonia and acute bronchitis. Macrolides, fluoroquinolones (FQs), and, recently, telithromycin (TEL) constitute primary therapeutic options, and rare cases of resistance have been reported. In this report, we describe the emergence of an
S. pneumoniae
clinical isolate with high-level TEL resistance (MIC, 256 μg/ml) and simultaneous resistance to FQs. Ongoing studies are oriented to elucidate the precise mechanism of resistance to TEL.
SELF-DUAL YANG–MILLS THEORY AS A SYSTEM FOR GENERATING INTEGRABLE SYSTEMS BY MEANS OF REDUCTIONS
Authors: BRACKEN PAUL
Published: November 2005

Self-dual Yang–Mills theory is presented and the associated equations are derived. An isospectral linear problem whose compatibility condition yields the self-dual Yang–Mills system is formulated. A characterization of the system in terms of a Lie-algebra valued current is given. Finally, several explicit reductions to specific integrable systems are derived.