bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTSerological 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

ABSTRACT

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

ABSTRACT
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.