bluuebunny/crossref_metadata_2025_split huggingface dataset:
Agrobacterium tumefaciens
Integrates Transfer DNA into Single Chromosomal Sites of Dimorphic Fungi and Yields Homokaryotic Progeny from Multinucleate Yeast
Authors: Sullivan Thomas D. , Rooney Peggy J. , Klein Bruce S. 
Published: December 2002

ABSTRACT

The dimorphic fungi
Blastomyces dermatitidis
and
Histoplasma capsulatum
cause systemic mycoses in humans and other animals. Forward genetic approaches to generating and screening mutants for biologically important phenotypes have been underutilized for these pathogens. The plant-transforming bacterium
Agrobacterium tumefaciens
was tested to determine whether it could transform these fungi and if the fate of transforming DNA was suited for use as an insertional mutagen. Yeast cells from both fungi and germinating conidia from
B. dermatitidis
were transformed via
A. tumefaciens
by using hygromycin resistance for selection. Transformation frequencies up to 1 per 100 yeast cells were obtained at high effector-to-target ratios of 3,000:1.
B. dermatitidis
and
H. capsulatum ura5
lines were complemented with transfer DNA vectors expressing
URA5
at efficiencies 5 to 10 times greater than those obtained using hygromycin selection. Southern blot analyses indicated that in 80% of transformants the transferred DNA was integrated into chromosomal DNA at single, unique sites in the genome. Progeny of
B. dermatitidis
transformants unexpectedly showed that a single round of colony growth under hygromycin selection or visible selection of transformants by
lacZ
expression generated homokaryotic progeny from multinucleate yeast. Theoretical analysis of random organelle sorting suggests that the majority of
B. dermatitidis
cells would be homokaryons after the ca. 20 generations necessary for colony formation. Taken together, the results demonstrate that
A. tumefaciens
efficiently transfers DNA into
B. dermatitidis
and
H. capsulatum
and has the properties necessary for use as an insertional mutagen in these fungi.
Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect
Authors: Schreiner Thomas , Mohrs Martina R. , Blau-Wasser Rosemarie , von Krempelhuber Alfred , Steinert Michael , Schleicher Michael , Noegel Angelika A. 
Published: December 2002

ABSTRACT

Comitin is an F-actin binding and membrane-associated protein from
Dictyostelium discoideum
, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of
Saccharomyces cerevisiae
particles and of
Escherichia coli
, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.
A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System
Authors: Mello Kerrianne , Daly Thomas M. , Morrisey Joanne , Vaidya Akhil B. , Long Carole A. , Bergman Lawrence W. 
Published: December 2002

ABSTRACTMerozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasitePlasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is theP. yoeliihomologue of MSP-7 originally described inPlasmodium falciparum. BLAST analysis of theP. falciparumdatabase has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. InP. falciparum,we designated these molecules MSRP-1 to -5. Analysis of theP. yoeliidatabase indicates a similar chromosomal organization for the two genes in the mouse parasite species. The threeP. falciparumsequences with the highest degree of homology to theP. yoeliisequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region ofP. falciparumMSP-1.
Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in
Plasmodium falciparum
-Infected Red Blood Cells
Authors: Kaviratne M. , Khan S. M. , Jarra W. , Preiser P. R. 
Published: December 2002

ABSTRACT

Malaria parasite antigens encoded by multigene families are important factors in virulence and in disease pathology. In
Plasmodium falciparum
, the virulence factor PfEMP-1 is encoded by the
var
multigene family and is exposed at the infected erythrocyte surface. PfEMP-1 is clonally variant, allowing the parasite to evade host immunity. The recently identified
P. falciparum stevor
multigene family and its products also have the potential to be involved in similar important aspects of host-parasite interactions. Here, we show tightly regulated stage-specific transcription of
stevor
occurring over just a few hours of the asexual parasite life cycle. Only a subset of
stevor
genes are transcribed in parasite populations maintained in cultures and in single micromanipulated parasites. Antibodies against STEVOR recognize proteins of the expected size (∼37 kDa) and localize STEVOR in Maurer's clefts, unique membranous structures located in the cytoplasm of infected erythrocytes. The fact that the timing of
stevor
expression and the location of STEVOR are clearly distinct from those of other parasite variant antigens suggests that this gene family may have a novel role in
P. falciparum
biology.
Inhibition of HSP90 in
Trypanosoma cruzi
Induces a Stress Response but No Stage Differentiation
Authors: Graefe Sebastian E. B. , Wiesgigl Martina , Gaworski Iris , Macdonald Andrea , Clos Joachim 
Published: December 2002

ABSTRACT

The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite
Leishmania donovani
. We investigated the role of HSP90 in the related parasite
Trypanosoma cruzi
by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G
1
phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in
T. cruzi
and serves as a feedback inhibitor in the cellular stress response. In contrast to
L. donovani
cells, however,
T. cruzi
cells treated with GA do not begin to differentiate into relevant life cycle stages.
Chromosome Localization Changes in the
Trypanosoma cruzi
Nucleus
Authors: Elias M. Carolina Q. B. , Faria Marcella , Mortara Renato A. , Motta Maria Cristina M. , de Souza Wanderley , Thiry Marc , Schenkman Sergio 
Published: December 2002

ABSTRACT

Chromosome localization in the interphase nuclei of eukaryotes depends on gene replication and transcription. Little is known about chromosome localization in protozoan parasites such as trypanosomes, which have unique mechanisms for the control of gene expression, with most genes being posttranscriptionally regulated. In the present study, we examined where the chromosomes are replicated in
Trypanosoma cruzi
, the agent of Chagas' disease. The replication sites, identified by the incorporation of 5-bromodeoxyuridine, are located at the nuclear periphery in proliferating epimastigote forms in the early S phase of the cell cycle. When the S phase ends and cells progress through the cell cycle, 5-bromodeoxyuridine labeling is observed in the nuclear interior, suggesting that chromosomes move. We next monitored chromosome locations in different stages of the cell cycle by using a satellite DNA sequence as a probe in a fluorescence in situ hybridization assay. We found two distinct labeling patterns according to the cell cycle stage. The first one is seen in the G
1
phase, in hydroxyurea-arrested epimastigotes or in trypomastigotes, which are differentiated nondividing forms. In all of these forms the satellite DNA is found in dots randomly dispersed in the nucleus. The other pattern is found in cells from the S phase to the G
2
phase. In these cells, the satellite DNA is found preferentially at the nuclear periphery. The labeling at the nuclear periphery disappears only after mitosis. Also, DNA detected with terminal deoxynucleotidyl transferase is found distributed throughout the nuclear space in the G
1
phase but concentrated at the nuclear periphery in the S phase to the G
2
phase. These results strongly suggest that
T. cruzi
chromosomes move and, after entering the S phase, become constrained at the nuclear periphery, where replication occurs.
ras2
Controls Morphogenesis, Pheromone Response, and Pathogenicity in the Fungal Pathogen
Ustilago maydis
Authors: Lee Nancy , Kronstad James W. 
Published: December 2002

ABSTRACT

Ustilago maydis
, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in
U. maydis
. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the
ras2
gene, which is predicted to encode a member of the well-conserved
ras
family of small GTP-binding proteins. Deletion of the
ras2
gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated
ras2
allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.
Analysis of Telomerase in
Candida albicans
: Potential Role in Telomere End Protection
Authors: Singh Sunitha M. , Steinberg-Neifach Olga , Mian I. Saira , Lue Neal F. 
Published: December 2002

ABSTRACT

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Analysis of the telomerase complex in the budding yeast
Saccharomyces cerevisiae
has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The genome of the pathogenic yeast
Candida albicans
contains putative orthologues of all three telomerase components. Disruption of each homologue resulted in significant but distinct telomere dysfunction in
Candida
. Similar to
S. cerevisiae
, the
Candida EST3
disruption strain exhibits progressive telomere loss over many generations, at a rate that is consistent with incomplete replication. In contrast, telomeres in both the
Candida TERT
and
EST1
disruption strains can contract rapidly, followed by partial or nearly complete recovery, suggesting a defect in telomere “capping.” We propose that these two telomerase subunits may participate in the protection of chromosomal ends in
Candida
. Analysis of telomerase-mediated primer extension in vitro indicates that only the TERT protein is absolutely essential for enzyme activity. Our results support the conservation of telomerase protein components beyond the catalytic subunit but reveal species-specific phenotypic alterations in response to loss of individual telomerase component. We also identify potential homologues of Est1p in phylogenetically diverse organisms. The Est1p sequence family possesses a conserved N-terminal domain predicted to be structurally related to tetratricopeptide repeat-containing proteins.
Loss of Compartmentalization Causes Misregulation of Lysine Biosynthesis in Peroxisome-Deficient Yeast Cells
Authors: Breitling Rainer , Sharif Orzala , Hartman Michelle L. , Krisans Skaidrite K. 
Published: December 2002

ABSTRACTTo characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data. Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes. Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected. We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation. We provide data that suggest that this is the result of a “pathological” stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde. Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts. This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome).
Multiple Functions of
mfa-1
, a Putative Pheromone Precursor Gene of
Neurospora crassa
Authors: Kim Hyojeong , Metzenberg Robert L. , Nelson Mary Anne 
Published: December 2002

ABSTRACT

A putative pheromone precursor gene of
Neurospora crassa
,
mfa-1
(which encodes mating factor
a
-1), was identified as the most abundant clone in starved mycelial and perithecial cDNA libraries. Northern analysis demonstrated high
mfa-1
expression in all mating type
a
tissues and suggested low expression levels in
mat A
tissues. The
mfa-1
gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3′ untranslated region (3′ UTR). The predicted MFA1 sequence showed 100% sequence identity to PPG2 of
Sordaria macrospora
and structural similarity (a carboxy-terminal CAAX motif) to many hydrophobic fungal pheromone precursors. Mutants with a disrupted open reading frame (ORF) in which the critical cysteine residue had been changed to a nonprenylatable residue, tyrosine (YAAX mutants), were isolated, as were
mfa-1
mutants with intact ORFs but multiple mutations in the 3′ noncoding region (CAAX mutants). The 3′ UTR is required for the full range of
mfa-1
gene activity. Both classes of mutants showed delayed and reduced vegetative growth (which was suppressed by supplementation with a minute amount [30 μM] of ornithine, citrulline, or arginine), as well as aberrant sexual development. When crossed as female parents to wild-type males, the CAAX and YAAX mutants showed greatly reduced ascospore production. No ascospores were produced in homozygous
mfa-1
crosses. As males, YAAX
mat a
mutants were unable to attract wild-type
mat A
trichogynes (female-specific hyphae) or to initiate sexual development, while CAAX
mat a
mutants were able to mate and produce sexual progeny despite their inability to attract
mat A
trichogynes. In the
mat A
background, both CAAX and YAAX mutants showed normal male fertility but defective vegetative growth and aberrant female sexual development. Thus, the
mfa-1
gene appears to have multiple roles in
N. crassa
development: (i) it encodes a hydrophobic pheromone with a putative farnesylated and carboxymethylated C-terminal cysteine residue, required by
mat a
to attract trichogynes of
mat A
; (ii) it is involved in female sexual development and ascospore production in both mating types; and (iii) it functions in vegetative growth of both mating types.
Reproductive tourism as moral pluralism in motion
Authors: Pennings G 
Published: December 2002

Reproductive tourism is the travelling by candidate service recipients from one institution, jurisdiction, or country where treatment is not available to another institution, jurisdiction, or country where they can obtain the kind of medically assisted reproduction they desire. The more widespread this phenomenon, the louder the call for international measures to stop these movements.Three possible solutions are discussed: internal moral pluralism, coerced conformity, and international harmonisation. The position is defended that allowing reproductive tourism is a form of tolerance that prevents the frontal clash between the majority who imposes its view and the minority who claim to have a moral right to some medical service. Reproductive tourism is moral pluralism realised by moving across legal borders. As such, this pragmatic solution presupposes legal diversity.
Palliative care research: trading ethics for an evidence base
Authors: Jubb A M 
Published: December 2002

Good medical practice requires evidence of effectiveness to address deficits in care, strive for further improvements, and justly apportion finite resources. Nevertheless, the potential of palliative care is still held back by a paucity of good evidence. These circumstances are largely attributable to perceived ethical challenges that allegedly distinguish dying patients as a special client class. In addition, practical limitations compromise the quality of evidence that can be obtained from empirical research on terminally ill subjects.This critique aims to appraise the need for focused research, in order to develop clinical and policy decisions that will guide health care professionals in their care of dying patients. Weighted against this need are tenets that value the practical and ethical challenges of palliative care research as unique and insurmountable. The review concludes that, provided investigators compassionately apply ethical principles to their work, there is no justification for not endeavouring to improve the quality of palliative care through research.
Withholding life prolonging treatment, and self deception
Authors: Sayers G M , Perera S 
Published: December 2002

Objectives:To compare non-treatment decision making by general practitioners and geriatricians in response to vignettes. To see whether the doctors’ decisions were informed by ethical or legal reasoning.Design:Qualitative study in which consultant geriatricians and general practitioners (GPs) randomly selected from a list of local practitioners were interviewed. The doctors were asked whether patients described in five vignettes should be admitted to hospital for further care, and to give supporting reasons. They were asked with whom they would consult, who they believed ought to make such decisions, and whether the relatives’ preferences would influence their decision making.Main measures:To analyse the factors influencing the doctors’ decisions not to admit otherwise terminally ill patients to hospital for life prolonging treatment.Results:Seventeen GPs and 18 geriatricians completed the interview. All vignettes produced strong concordance in decision making between both groups. Ten per cent of the doctors would provide life prolonging treatment to patients with severe brain damage. Most would admit a surgical patient regardless of age or disability. Medical reasons were largely used to explain decision making. The wishes of relatives were influential and resource considerations were not. There was variability regarding decision making responsibility.Conclusions:Little attempt was made to link decision making with ethical or legal concepts and there may have been non-recognition, or denial, of the ethical consequences of failure to admit. The process of decision making may involve deception. This may be conscious, because of the illegality of euthanasia, or unconscious (self deception), because of deepseated medical and societal reluctance to accept that intentionally withholding life prolonging treatment may equate with intentionally causing death.
Evaluation of do not resuscitate orders (DNR) in a Swiss community hospital
Authors: Junod Perron N , Morabia A , de Torrenté A 
Published: December 2002

Objective:To evaluate the effect of an intervention on the understanding and use of DNR orders by physicians; to assess the impact of understanding the importance of involving competent patients in DNR decisions.Design:Prospective clinical interventional study.Setting:Internal medicine department (70 beds) of the hospital of La Chaux-de-Fonds, Switzerland.Participants:Nine junior physicians in postgraduate training.Intervention:Information on the ethics of DNR and implementation of new DNR orders.Measurements and main results:Accurate understanding, interpretation, and use of DNR orders, especially with respect to the patients’ involvement in the decision were measured. Junior doctors writing DNR orders had an extremely poor understanding of what DNR orders mean. The correct understanding of the definition of a DNR order increased from 31 to 93% (p<0.01) after the intervention and the patients’ involvement went from 17% to 48% (p<0.01). Physicians estimated that 75% of their DNR patients were mentally competent at the time of the decision.Conclusion:An intervention aimed at explaining the ethical principles and the definition of DNR orders improves understanding of them, and their implementation, as well as patient participation. Specific efforts are needed to increase the involvement of mentally competent patients in the decision.