bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of a Brief Nurse‐Administered Migraine Assessment Tool
Authors: Marcus Dawn A. , Kapelewski Cindy , Jacob Rolf G. , Rudy Thomas E. , Furman Joseph M.
Published: April 2004

Objective.—To validate a brief tool for screening migraine.

Background.—Migraine is a common, but underdiagnosed condition. Effective utilization of nonphysician personnel to reliably screen patients for migraine may improve identification of migraineurs for clinical treatment and research.

Methods.—An 8‐question Migraine Assessment Tool (based on International Headache Society criteria) was designed for administration by a nurse with no specialized headache training as a pre‐assessment for the diagnosis of migraine for use in either a research or clinical environment. A community sample of 80 adults (71 women, 9 men; mean age, 33.7 years; 80% white, 14% African American, 2.5% Asian American) with self‐reported headache was recruited through advertisements. A headache specialist independently diagnosed subjects using clinical assessment, and a nurse who works in a balance disorder clinic used the Migraine Assessment Tool. Agreement between physician and nurse‐administered Migraine Assessment Tool diagnoses was determined. Each subject returned in 2 to 4 weeks for a second assessment, administered by the same nurse. Agreement between the 2 diagnoses from the Migraine Assessment Tool was calculated.

Results.—Comparison between diagnosis by the physician versus the Migraine Assessment Tool revealed a positive predictive value of 0.85; negative predictive value, 0.84; sensitivity, 0.89; specificity, 0.79; and observed agreement, 0.85. Cohen's kappa reliability measure was 0.69, indicating good test reliability. Interestingly, in 8 of the 12 cases of disagreement, the examiner diagnosing nonmigraine diagnosed analgesic overuse headache. Comparing diagnoses assigned by the 2 separate administrations of the Migraine Assessment Tool revealed a Cohen's kappa of 0.69. Notably, 9 of the 12 cases of nonagreement on the 2 assessments were due to subjects endorsing analgesic overuse in only 1 of the 2 testing sessions.

Conclusions.—This study showed good reliability and stability of a new, brief, nurse‐administered migraine questionnaire. In addition, the study also showed that consistency in self‐reporting analgesic overuse within individuals with headache is poor. This suggests the need for repeat questioning about analgesic overuse on subsequent appointments to ensure absence of analgesic overuse headache.
Short‐Period Heart Rate Variability in the General Population as Compared to Patients with Acute Myocardial Infarction from the Same Source Population
Authors: Kuch Bernhard , Parvanov Toma , Hense Hans W. , Axmann Joerg , Bolte Heinz D.
Published: April 2004

Background: Our aim was to compare the distribution and determinants of heart rate variability (HRV) measures in a middle‐aged population with patients of the same sex and age after an acute myocardial infarction (AMI), and to show, whether HRV values defined as abnormal from the general population are indicative for a worse prognosis even in AMI patients.

Methods: HRV was studied in a random sample of 149 middle‐aged men and 137 women from the general population (45–65 years) as well as 129 consecutive AMI patients (25–74 years). Spectral analysis was used to compute low frequency (LF), high frequency (HF), and total frequency power. To the AMI population of age 45–65 years (N = 85) a sample out of the general population was matched by age and sex by 2:1 matching (N = 149). All AMI patients were followed for a median of 43 months (range 1–47) for death or malignant arrhythmia.

Results: All measures of HRV were significantly and substantially lower in AMI patients than the general population (P < 0.001). Expression in relative terms revealed that the proportionate contributions of HF and LF to total power were significantly different in the two populations with relatively lower LF power in AMI patients (P < 0.01). The negative correlation with heart rate and HRV measures was significantly more pronounced in AMI patients (P < 0.01). The 2.5th percentile of the LF power distribution in the general population (3.08 ln ms2) corresponds to the 25th percentile in the AMI population. Subjects of the whole AMI population with values below this LF cutpoint revealed a significant increased risk of death or malignant arrhythmia during follow‐up (odds ratio 5.1; 95% confidence interval: 1.3; 23).

Conclusions: AMI patients had strongly diminished HRV compared to the general population. The relatively lower LF power indicates an alteration of the sympathico‐vagal balance, and the significantly stronger correlation of heart rate with HRV may be indicative for a more pronounced effect of sympathetic activation on autonomic modulation in the case of myocardial infarction. Finally, a value below the 2.5th percentile of the population LF power distribution may identify subjects at risk and warrant further testing.
Cardiac Autonomic Modulation by Estrogen in Female Mice Undergoing Ambulatory Monitoring and In Vivo Electrophysiologic Testing
Authors: Saba Samir , Shusterman Vladimir , Usiene Irmute , London Barry
Published: April 2004

Introduction: Estrogen is an important modulator of cardiovascular risk, but its mechanism of action is not fully understood. We investigated the effect of ovariectomy and its timing on the cardiac electrophysiology in mice.

Methods: Thirty female mice (age 18.8 ± 3.1 weeks) underwent in vivo electrophysiologic testing before and after autonomic blockade. Fifteen mice were ovariectomized prepuberty (PRE) and ten postpuberty (POST), 2 weeks prior to electrophysiologic testing. Five age‐matched sham‐operated female mice (Control) served as controls. A subset of 13 mice (5 PRE, 3 POST, and 5 Controls) underwent 24‐hour ambulatory monitoring.

Results: With ambulatory monitoring, the average (668 ± 28 vs 769 ± 52 b/min, P = 0.008) and minimum (485 ± 47 vs 587 ± 53 b/min, P = 0.02) heart rates were significantly slower in the ovariectomized mice (PRE and POST groups) compared to the Control group. At baseline electrophysiologic testing, there were no significant differences among the ovariectomized and intact mice in any of the measured parameters. With autonomic blockade, the Control group had a significantly larger change (▵) in the atrioventricular (AV) nodal Wenckebach (AVW) periodicity (▵AVW = 11.3 ± 2.9 vs 2.1 ± 7.3 ms, P = 0.05) and functional refractory period (▵FRP = 11.3 ± 2.1 vs 1.25 ± 6.8 ms, P = 0.02) compared to the ovariectomized mice. These results were not altered by the time of ovariectomy (PRE vs POST groups).

Conclusion: Our results suggest that estrogen modulates the autonomic inputs into the murine sinus and AV nodes. These findings, if replicated in humans, might underlie the observed clustering of certain arrhythmias around menstruation and explain the higher incidence of arrhythmias in men and postmenopausal women.
Sex Steroids and Heart Rate Variability in Patients after Myocardial Infarction
Authors: Wranicz Jerzy Krzysztof , Rosiak Marcin , Cygankiewicz Iwona , Kula Piotr , Kula Krzysztof , Zareba Wojciech
Published: April 2004

Background: Although the relationship between sex steroid levels and coronary artery disease (CAD) has been the subject of many studies there are still controversies concerning the role of sex steroids in CAD. In patients with CAD, especially after a myocardial infarction, there is evidence for autonomic nervous system dysfunction. However, there is no data detailing the relationship between sex steroids and cicardian autonomic activity in patients with CAD. The aim of the study was to evaluate the association between sex steroids and heart rate variability (HRV) parameters in postinfarction patients.

Methods: In 88 postinfarction men (aged 36–73, average 53 years), 24‐hour Holter monitoring was performed to assess HRV parameters: SDNN, SDNNI, SDANN, rMSSD, pNN50, and levels of the following hormones were measured: testosterone, estradiol, free testosterone index, and estradiol/testosterone ratio. Univariate and multivariate regression analyses were used to investigate the relationship between HRV parameters and levels of tested hormones.

Results: Increased testosterone levels were associated with increased SDNN (r = 0.38, P = 0.03), increased rMSSD (r = 0.51, P = 0.002), and increased pNN50 (r = 0.45, P = 0.007). These associations remained significance after adjustment for age, ejection fraction, and other relevant clinical covariates. There was no significant association between estradiol and HRV parameters.

Conclusion: In men with a history of myocardial infarction, higher levels of testosterone are associated with higher HRV measures of parasympathetic activity. These findings suggest that testosterone beneficially influences autonomic regulation of the heart.
A preliminary investigation into the ultrastructure of dental calculus and associated bacteria
Authors: Tan B. , Gillam David G. , Mordan N. J. , Galgut P. N.
Published: April 2004

Introduction: Though dental calculus is generally recognised as comprising mineralised bacteria, areas of non‐mineralised bacteria may be present.

Aim: To investigate the ultrastructure of non‐decalcified young and mature supragingival calculus and subgingival calculus, and the possible presence of internal viable bacteria.

Materials and methods: Supragingival calculus was harvested from five patients, 9–10 weeks after scaling and root debridement. Five samples of mature supragingival and subgingival calculus were taken from patients presenting with adult periodontitis. Specimens were fixed and embedded for transmission electron microscopy.

Results: The ultrastructure of young and mature supragingival calculus was similar with various large and small crystal types. Non‐mineralised channels were observed extending into the calculus, often joining extensive lacunae, both containing intact non‐mineralised coccoid and rod‐shaped microorganisms. Subgingival calculus possessed more uniform mineralisation without non‐mineralised channels and lacunae.

Conclusion: Supragingival calculus contains non‐mineralised areas which contain bacteria and other debris. The viability of the bacteria, and their identification could not be determined in this preliminary investigation. As viable bacteria within these lacunae may provide a source of re‐infection, further work needs to be done to identify the bacteria in the lacunae, and to determine their viability.
Structure‐Function Analysis of the BEACH Protein LvsA
Authors: Wu Wei‐I , Yajnik Juhi , Siano Michael , De Lozanne Arturo
Published: April 2004

Most eukaryotes have several members of the BEACH family of proteins but the molecular function of these large proteins remains unknown. The Dictyostelium BEACH protein LvsA is essential for cytokinesis and contractile vacuole activity. The functional contribution of different portions of LvsA was tested here by deletion analysis. The C‐terminal WD domain was important for protein stability and C‐terminal deletions resulted in loss of LvsA function. In contrast, N‐terminal deletions yielded abundant protein expression that could be assayed for function. Despite very low sequence conservation of the N‐terminal portion of LvsA, this region is important for its function in vivo. Deletion of 689 N‐terminal amino acids produced a protein that was functional in cytokinesis but partially functional in osmoregulation. Further deletions resulted in the complete loss of LvsA function. Using in vitro fractionation assays we found that LvsA sedimented with membranes but that this association does not require the N‐terminal portion of LvsA. Interestingly, the association of LvsA with the contractile vacuole was perturbed by the loss of drainin, a protein important for vacuole function. In drainin‐null cells, LvsA bound irreversibly to engorged contractile vacuoles that fail to expel water. These experiments help delineate the biochemical and physiological requirements for function of one important BEACH protein, LvsA.
Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a
Trypanosoma brucei
Glycosylation Mutant
Authors: Acosta-Serrano Alvaro , O'Rear Jessica , Quellhorst George , Lee Soo Hee , Hwa Kuo-Yuan , Krag Sharon S. , Englund Paul T.
Published: April 2004

Concanavalin A (ConA) kills the procyclic (insect) form of
Trypanosoma brucei
by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [
3
H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man
9
GlcNAc
2
, whereas that from ConA 1-1 is Man
7
GlcNAc
2
. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man
4
GlcNAc
2
modified with a terminal
N
-acetyllactosamine group, which is poorly recognized by ConA.
Plasmodium falciparum
Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling
Authors: Fan Qi , An Lijia , Cui Liwang
Published: April 2004

The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in
Plasmodium
species and that the most homologous regions are within the HAT domain and the bromodomain. The
Plasmodium falciparum GCN5
homologue (
PfGCN5
) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the
PfGCN5
transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in
P. falciparum
, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the
yGCN5
deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the
yGCN5
deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in
P. falciparum
.
YBP1 and Its Homologue YBP2/YBH1 Influence Oxidative-Stress Tolerance by Nonidentical Mechanisms in
Saccharomyces cerevisiae
Authors: Gulshan Kailash , Rovinsky Sherry A. , Moye-Rowley W. Scott
Published: April 2004

In the yeast
Saccharomyces cerevisiae
, the transcription factor Yap1p is a central determinant of resistance to oxidative stress. Previous work has demonstrated that Yap1p is recruited from the cytoplasm to the nucleus upon exposure to the oxidants diamide and H
2
O
2
in a process that requires the transient covalent linkage of the glutathione peroxidase Gpx3p to Yap1p. Genetic and biochemical analyses indicate that while both oxidants trigger nuclear accumulation of Yap1p, the function and regulation of this transcription factor is different under these two different oxidative stresses. Ybp1p (Yap1p-binding protein) has recently been demonstrated to be required for Yap1p-mediated H
2
O
2
resistance but not diamide resistance. A Ybp1p homologous protein (Ybh1p/Ybp2p) was also detected in the
S. cerevisiae
genome. Here we compare the actions of these two closely related proteins and provide evidence that while both factors influence H
2
O
2
tolerance, they do so by nonidentical mechanisms. A double mutant strain lacking both
YBP1
and
YBH1
genes is more sensitive to H
2
O
2
and more defective in activation of Yap1p-dependent gene expression than either single mutant. Ybp1p has a more pronounced effect on these phenotypes than does Ybh1p. Protein-protein interactions between Yap1p and Ybp1p could be detected by either the yeast two-hybrid or coimmunoprecipitation approach while neither technique could demonstrate Yap1p-Ybh1p interactions. Overexpression experiments indicated that high levels of Ybh1p but not Ybp1p could bypass the H
2
O
2
hypersensitivity of a
gpx3
Δ strain. Together, these data argue that these two homologous proteins act as parallel positive regulators of H
2
O
2
tolerance.
Identification, Mutational Analysis, and Coactivator Requirements of Two Distinct Transcriptional Activation Domains of the
Saccharomyces cerevisiae
Hap4 Protein
Authors: Stebbins John L. , Triezenberg Steven J.
Published: April 2004

The Hap4 protein of the budding yeast
Saccharomyces cerevisiae
activates the transcription of genes that are required for growth on nonfermentable carbon sources. Previous reports suggested the presence of a transcriptional activation domain within the carboxyl-terminal half of Hap4 that can function in the absence of Gcn5, a transcriptional coactivator protein and histone acetyltransferase. The boundaries of this activation domain were further defined to a region encompassing amino acids 359 to 476. Within this region, several clusters of hydrophobic amino acids are critical for transcriptional activity. This activity does not require
GCN5
or two other components of the SAGA coactivator complex,
SPT3
and
SPT8
, but it does require
SPT7
and
SPT20
. Contrary to previous reports, a Hap4 fragment comprising amino acids 1 to 330 can support the growth of yeast on lactate medium, and when tethered to
lexA
, can activate a reporter gene with upstream
lexA
binding sites, demonstrating the presence of a second transcriptional activation domain. In contrast to the C-terminal activation domain, the transcriptional activity of this N-terminal region depends on
GCN5
. We conclude that the yeast Hap4 protein has at least two transcriptional activation domains with strikingly different levels of dependence on specific transcriptional coactivator proteins.
Novel P-Type ATPases Mediate High-Affinity Potassium or Sodium Uptake in Fungi
Authors: Benito Begoña , Garciadeblás Blanca , Schreier Peter , Rodríguez-Navarro Alonso
Published: April 2004

Fungi have an absolute requirement for K+, but K+may be partially replaced by Na+. Na+uptake inUstilago maydisandPichia sorbitophilawas found to exhibit a fast rate, lowKm, and apparent independence of the membrane potential. Searches of sequences with similarity to P-type ATPases in databases allowed us to identify three genes in these species,Umacu1,Umacu2, andPsACU1, that could encode P-type ATPases of a novel type. Deletion of theacu1andacu2genes proved that they encoded the transporters that mediated the high-affinity Na+uptake ofU. maydis. Heterologous expressions of theUmacu2gene in K+transport mutants ofSaccharomyces cerevisiaeand transport studies in the single and double Δacu1and Δacu2mutants ofU. maydisrevealed that theacu1andacu2genes encode transporters that mediated high-affinity K+uptake in addition to Na+uptake. Other fungi also have genes or pseudogenes whose translated sequences show high similarity to the ACU proteins ofU. maydisandP. sorbitophila. In the phylogenetic tree of P-type ATPases all the identified ACU ATPases define a new cluster, which shows the lowest divergence with type IIC, animal Na+,K+-ATPases. The fungal high-affinity Na+uptake mediated by ACU ATPases is functionally identical to the uptake that is mediated by some plant HKT transporters.
Cryptococcus neoformans CAP59
(or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan
Authors: García-Rivera Javier , Chang Yun C. , Kwon-Chung K. J. , Casadevall Arturo
Published: April 2004

Several genes are essential for
Cryptococcus neoformans
capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in
cap59
mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in
CAP59
, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The
cap59
gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the
cap59
deletant, assimilated raffinose or urea. Hence, the missense mutation of
CAP59
in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that
CAP59
is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.
A Paradoxical Mutant GATA Factor
Authors: Muro-Pastor M. Isabel , Strauss Joseph , Ramón Ana , Scazzocchio Claudio
Published: April 2004

The
niiA
(nitrite reductase) and
niaD
(nitrate reductase) genes of
Aspergillus nidulans
are subject to both induction by nitrate and repression by ammonium or glutamine. The intergenic region between these genes functions as a bidirectional promoter. In this region, nucleosomes are positioned under nonexpression conditions. On nitrate induction under derepressing conditions, total loss of positioning occurs. This is independent of transcription and of the NirA-specific transcription factor but absolutely dependent on the wide-domain GATA-binding AreA factor. We show here that a 3-amino-acid deletion in the basic carboxy-terminal sequence of the DNA-binding domain results in a protein with paradoxical properties. Its weak DNA binding is consistent with its loss-of-function phenotype on most nitrogen sources. However, it results in constitutive expression and superinducibility of
niiA
and
niaD
. Nucleosome loss of positioning is also constitutive. The mutation partially suppresses null mutations in the transcription factor NirA. AreA binds NirA in vitro, and the mutation does not affect this interaction. The in vivo methylation pattern of the promoter is drastically altered, suggesting the recruitment of one or more unknown transcription factors and/or a local distortion on the DNA double helix.
Cell Division Defects of
Schizosaccharomyces pombe liz1
−
Mutants Are Caused by Defects in Pantothenate Uptake
Authors: Stolz Jürgen , Caspari Thomas , Carr Antony M. , Sauer Norbert
Published: April 2004

The

liz1
+

gene of the fission yeast
Schizosaccharomyces pombe
was previously identified by complementation of a mutation that causes abnormal mitosis when ribonucleotide reductase is inhibited. Liz1 has similarity to transport proteins from
Saccharomyces cerevisiae
, but the potential substrate and its connection to the cell division cycle remain elusive. We report here that

liz1
+

encodes a plasma membrane-localized active transport protein for the vitamin pantothenate, the precursor of coenzyme A (CoA). Liz1 is required for pantothenate uptake at low extracellular concentrations. A lack of pantothenate uptake results in three phenotypes: (i) slow growth, (ii) delayed septation, and (iii) aberrant mitosis in the presence of hydroxyurea (HU). All three phenotypes are suppressed by high extracellular concentrations of pantothenate, where pantothenate uptake occurs by passive diffusion.
liz1
Δ mutants are viable because they can synthesize pantothenate from uracil as an endogenous source. The use of uracil for both pantothenate biosynthesis and deoxyribonucleotide generation provides an explanation for the aberrant mitosis in the presence of HU. HU blocks ribonucleotide reductase, and we propose that the accumulation of ribonucleotides reduces uracil biosynthesis by feedback inhibition of aspartate transcarbamoylase. Thus, the addition of HU to
liz1
Δ mutants results in a shortage of pantothenate. Because
liz1
Δ mutants show striking similarities to mutants with defects in fatty acid biosynthesis, we propose that the shortage of pantothenate compromises fatty acid synthesis, resulting in slow growth and mitotic defects.
Challenge of
Drosophila melanogaster
with
Cryptococcus neoformans
and Role of the Innate Immune Response
Authors: Apidianakis Yiorgos , Rahme Laurence G. , Heitman Joseph , Ausubel Frederick M. , Calderwood Stephen B. , Mylonakis Eleftherios
Published: April 2004

We found that the ingestion of
Cryptococcus neoformans
by
Drosophila melanogaster
resulted in the death of the fly but that the ingestion of
Saccharomyces cerevisiae
or the nonpathogenic
Cryptococcus kuetzingii
or
Cryptococcus laurentii
did not. The
C. neoformans
protein kinase A and RAS signal transduction pathways, previously shown to be involved in virulence in mammals, also played a role in killing
Drosophila
. Mutation of the Toll immune response pathway, the predominant antifungal pathway of the fly, did not play a role in
Drosophila
defense following ingestion of the yeast. However, the Toll pathway was necessary for the clearance of
C. neoformans
introduced directly into the hemolymph of
D. melanogaster
and for the survival of systemically infected flies.
Cryptococcus neoformans
Virulence Gene Discovery through Insertional Mutagenesis
Authors: Idnurm Alexander , Reedy Jennifer L. , Nussbaum Jesse C. , Heitman Joseph
Published: April 2004

Insertional mutagenesis was applied to
Cryptococcus neoformans
to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (
NAT
)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The
NAT
insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene
FHB1
, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation,
Agrobacterium
-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to
NAT
in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the
LAC1
(laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the
CLC1
gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu
+
transport into the secretory pathway is compromised, depriving laccase and other Cu
+
-dependent proteins of their essential cofactor. The
NAT
resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but
Agrobacterium
-mediated transformation is more efficient and generates exclusively stable insertion mutations.
Cell Cycle Progression in G
1
and S Phases Is
CCR4
Dependent following Ionizing Radiation or Replication Stress in
Saccharomyces cerevisiae
Authors: Westmoreland Tammy J. , Marks Jeffrey R. , Olson John A. , Thompson Eric M. , Resnick Michael A. , Bennett Craig B.
Published: April 2004

To identify new nonessential genes that affect genome integrity, we completed a screening for diploid mutant
Saccharomyces cerevisiae
strains that are sensitive to ionizing radiation (IR) and found 62 new genes that confer resistance. Along with those previously reported (Bennett et al., Nat. Genet. 29:426-434, 2001), these genes bring to 169 the total number of new IR resistance genes identified. Through the use of existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4-NOT and RNA polymerase-associated factor 1 (PAF1)-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G
1
cell population is radiosensitive. Consistent with a role in G
1
, diploid
ccr4
Δ cells irradiated in G
1
show enhanced lethality compared to cells exposed as a synchronous G
2
population. In addition, a prolonged
RAD9
-dependent G
1
arrest occurred following IR of
ccr4
Δ cells and
CCR4
is a member of the
RAD9
epistasis group, thus confirming a role for
CCR4
in checkpoint control. Moreover,
ccr4
Δ cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G
2
followed by cellular lysis. This S-phase replication defect is separate from that seen for
rad52
mutants, since
rad52
Δ
ccr4
Δ cells show increased sensitivity to HU compared to
rad52
Δ or
ccr4
Δ mutants alone. These results indicate that cell cycle transition through G
1
and S phases is
CCR4
dependent following radiation or replication stress.
A Novel Allele of
Saccharomyces cerevisiae NDC1
Reveals a Potential Role for the Spindle Pole Body Component Ndc1p in Nuclear Pore Assembly
Authors: Lau Corine K. , Giddings Thomas H. , Winey Mark
Published: April 2004

Both the spindle pole body (SPB) and the nuclear pore complex (NPC) are essential organelles embedded in the nuclear envelope throughout the life cycle of the budding yeast
Saccharomyces cerevisiae
. However, the mechanism by which these two multisubunit structures are inserted into the nuclear envelope during their biogenesis is not well understood. We have previously shown that Ndc1p is the only known integral membrane protein that localizes to both the SPBs and the NPCs and is required for SPB duplication. For this study, we generated a novel temperature-sensitive (
ts
) allele of
NDC1
to investigate the role of Ndc1p at the NPCs. Yeast cells carrying this allele (
ndc1
-
39
) failed to insert the SPB into the nuclear envelope at the restrictive temperature. Importantly, the double mutation of
ndc1
-
39
and NPC assembly mutant
nic96
-
1
resulted in cells with enhanced growth defects. While nuclear protein import and NPC distribution in the nuclear envelope were unaffected,
ndc1
-
39
mutants failed to properly incorporate the nucleoporin Nup49p into NPCs. These results provide evidence that Ndc1p is required for NPC assembly in addition to its role in SPB duplication. We postulate that Ndc1p is crucial for the biogenesis of both the SPBs and the NPCs at the step of insertion into the nuclear envelope.
Tfs1p, a Member of the PEBP Family, Inhibits the Ira2p but Not the Ira1p Ras GTPase-Activating Protein in
Saccharomyces cerevisiae
Authors: Chautard Hélène , Jacquet Michel , Schoentgen Françoise , Bureaud Nicole , Bénédetti Hélène
Published: April 2004

Ras proteins are guanine nucleotide-binding proteins that are highly conserved among eukaryotes. They are involved in signal transduction pathways and are tightly regulated by two sets of antagonistic proteins: GTPase-activating proteins (GAPs) inhibit Ras proteins, whereas guanine exchange factors activate them. In this work, we describe Tfs1p, the first physiological inhibitor of a Ras GAP, Ira2p, in
Saccharomyces cerevisiae. TFS1
is a multicopy suppressor of the
cdc25-1
mutation in yeast and corresponds to the so-called Ic CPY cytoplasmic inhibitor. Moreover, Tfs1p belongs to the phosphatidylethanolamine-binding protein (PEBP) family, one member of which is RKIP, a kinase and serine protease inhibitor and a metastasis inhibitor in prostate cancer. In this work, the results of (i) a two-hybrid screen of a yeast genomic library, (ii) glutathione
S
-transferase pulldown experiments, (iii) multicopy suppressor tests of
cdc25-1
mutants, and (iv) stress resistance tests to evaluate the activation level of Ras demonstrate that Tfs1p interacts with and inhibits Ira2p. We further show that the conserved ligand-binding pocket of Tfs1—the hallmark of the PEBP family—is important for its inhibitory activity.
Transcription Initiation and Termination on
Leishmania major
Chromosome 3
Authors: Martínez-Calvillo Santiago , Nguyen Dan , Stuart Kenneth , Myler Peter J.
Published: April 2004

Genome projects involving
Leishmania
and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire
Leishmania major
Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the “left” telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the “right” telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.
Mitochondrial Histone-Like DNA-Binding Proteins Are Essential for Normal Cell Growth and Mitochondrial Function in
Crithidia fasciculata
Authors: Avliyakulov Nuraly K. , Lukeš Julius , Ray Dan S.
Published: April 2004

The
Crithidia fasciculata
KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The
KAP2
and
KAP3
genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire
KAP2
and
KAP3
locus increases maxicircle mRNA levels two- to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.
Candida albicans
Biofilms: a Developmental State Associated With Specific and Stable Gene Expression Patterns
Authors: García-Sánchez Susana , Aubert Sylvie , Iraqui Ismaïl , Janbon Guilhem , Ghigo Jean-Marc , d'Enfert Christophe
Published: April 2004

Like many bacteria, yeast species can form biofilms on several surfaces.
Candida albicans
colonizes the surfaces of catheters, prostheses, and epithelia, forming biofilms that are extremely resistant to antifungal drugs. We have used transcript profiling to investigate the specific properties of
C. albicans
biofilms. Biofilm and planktonic cultures produced under different conditions of nutrient flow, aerobiosis, or glucose concentration were compared by overall gene expression correlation. Correlation was much higher between biofilms than planktonic populations irrespective of the growth conditions, indicating that biofilm populations formed in different environments display very similar and specific transcript profiles. A first cluster of 325 differentially expressed genes was identified. In agreement with the overrepresentation of amino acid biosynthesis genes in this cluster, Gcn4p, a regulator of amino acid metabolism, was shown to be required for normal biofilm growth. To identify biofilm-related genes that are independent of mycelial development, we studied the transcriptome of biofilms produced by a wild-type, hypha-producing strain and a
cph1
/
cph1 efg1
/
efg1
strain defective for hypha production. This analysis identified a cluster of 317 genes expressed independently of hypha formation, whereas 86 genes were dependent on mycelial development. Both sets revealed the activation of the sulfur-amino acid biosynthesis pathway as a feature of
C. albicans
biofilms.
Dual-Color Imaging of Nuclear Division and Mitotic Spindle Elongation in Live Cells of
Aspergillus nidulans
Authors: Su Wenqi , Li Shihe , Oakley Berl R. , Xiang Xin
Published: April 2004

We have developed a dual-color imaging system based on cyan fluorescent protein-labeled histone H2A and green fluorescent protein-labeled alpha tubulin to visualize DNA and spindles simultaneously in the same living cell of
Aspergillus nidulans
. This system allows new details of mitosis and nuclear movement to be revealed.
A Mitogen-Activated Protein Kinase That Senses Nitrogen Regulates Conidial Germination and Growth in
Aspergillus fumigatus
Authors: Xue Tao , Nguyen C. Kim , Romans Angela , May Gregory S.
Published: April 2004

We show that the mitogen-activated protein (MAP) kinase pathway that responds to osmotic stress in
Aspergillus fumigatus
is also involved in nutritional sensing. This MAP kinase regulates conidial germination in response to the nitrogen source and is activated upon starvation for either carbon or nitrogen during vegetative growth.
Biosynthesis and Uptake of Siderophores Is Controlled by the PacC-Mediated Ambient-pH Regulatory System in
Aspergillus nidulans
Authors: Eisendle Martin , Oberegger Harald , Buttinger Rudolf , Illmer Paul , Haas Hubertus
Published: April 2004

Biosynthesis and uptake of siderophores in
Aspergillus nidulans
are regulated not only by iron availability but also by ambient pH: expression of this high-affinity iron uptake system is elevated by an increase in the ambient pH. Mediation of this regulation by the transcriptional regulator PacC has been confirmed via acidity- and alkalinity-mimicking mutants.
Identification of Genes Dependent on the MADS Box Transcription Factor SrfA in
Dictyostelium discoideum
Development
Authors: Escalante Ricardo , Iranfar Negin , Sastre Leandro , Loomis William F.
Published: April 2004

Analysis of microarrays containing 6,345
Dictyostelium discoideum
genes has identified 21 whose expression is dependent on the MADS box transcription factor SrfA. In wild-type cells, all of these genes are induced late in development. At least four of them are necessary for proper spore differentiation, stability, and/or germination.
Scurvy in the British Mercantile Marine in the 19th century, and the contribution of the Seamen’s Hospital Society
Authors: Cook G C
Published: April 2004

When long voyages in sailing vessels were commonplace, scurvy was a major health hazard in mariners of all nations. The observations of James Lind (1716–94) and others indicated that citrus fruits had both a preventive and curative role in this disease. In the light of this work, by 1800 the disease had been virtually eliminated from Britain’s Royal Navy. However, it continued in the merchant navies of all nations until the latter half of the 19th century. In 1867, the Merchant Shipping Amendment Act was passed by the British Parliament largely as a result of a concerted effort by the Seamen’s Hospital Society (SHS), one of whose physicians, Harry Leach (1836–79) was the major proselytiser for improved conditions in the merchant service. Examination of the SHS records before and after this event demonstrate a marked reduction in the prevalence of scurvy in the Port of London. Although other factors—such as the introduction of steam ships, which resulted in faster voyages—were clearly important, the compulsory administration of genuine lime juice under supervision in the merchant service seems to have exerted a significant effect.
Pneumocystis carinii pneumonia in chronic lymphocytic leukaemia
Authors: Vavricka S R , Halter J , Hechelhammer L , Himmelmann A
Published: April 2004

Pneumocystis carinii pneumonia in patients with chronic lymphocytic leukaemia (CLL) who have not been treated with fludarabin are rare, although clinically relevant CD4 T-cell depletion can occur in longstanding CLL without prior treatment with purine analogues. A 52 year old woman is reported who was on long term treatment with chlorambucil and taking a short course of prednisone for familial CLL before she developped progressive dyspnoea, and P carinii pneumonia was diagnosed in bronchoalveolar lavage fluid. Despite treatment with high dose co-trimoxazole the patient died.
Echocardiographic abnormalities in primary antibody deficiency
Authors: Johnston S L , Hill S J , Lock R J , Dwight J F , Unsworth D J , Gompels M M
Published: April 2004

Objective

To document cardiac abnormalities secondary to pulmonary disease in primary antibody deficiency.

Patients and methods

A cross sectional audit study of patients from a regional immunology centre. Subjects undergoing two dimensional and Doppler transthoracic echocardiography were reviewed. Ventricular dimensions and function, valvular competence, and estimated pulmonary artery pressure were recorded. Data were compared with clinical variables, pulmonary function tests, and thoracic computed tomography data.

Results

Nineteen patients with common variable immunodeficiency and one with IgG2 subclass deficiency were included, mean age at diagnosis 37.5 years, mean estimated diagnostic delay 10.94 years. Left ventricular impairment was found in 15% and right heart dilatation in 20%. Pulmonary hypertension (mean pulmonary artery pressure >25 mm Hg) was found in 45% (9/20), graded as moderate (40–60 mm Hg) in 44% of cases. Pulmonary function was obstructive in 47% (9/19). Fifty five percent of the patients with computed tomography data within the last five years (10/18) had confirmed bronchiectasis. Patients with right heart dilatation and/or moderate pulmonary hypertension (n = 6) had a more prolonged diagnostic delay (p = 0.04) and more severe lung disease.

Conclusion

Echocardiographic abnormalities are common in primary antibody deficiency, associated with diagnostic delay and pulmonary complications. Pulmonary hypertension should be considered in those with severe lung disease and can be confirmed by echocardiography.
Establishing the Diploma in Tropical Medicine and Hygiene (DTM&H) course in the Sultanate of Oman
Authors: Scrimgeour E M
Published: April 2004

Until 1997, there was no formal teaching in tropical medicine in Arabian Peninsular countries, although many tropical diseases (for example, malaria, schistosomiasis, leishmaniasis, filariasis), are endemic in the region. A six month, part time Diploma in Tropical Medicine and Hygiene (DTM&H) course was established in the Sultan Qaboos University, Muscat, Sultanate of Oman, in 1997, for small groups of Omani doctors wishing to sit the annual DTM&H examination in London. To date, 21 doctors have been successful, and the Royal College of Physicians of London has accredited the Tropical Medicine School in Oman as an appropriate training centre for the examination. Since over 20 doctors apply each year for the six available places in the course, a full time DTM&H course for larger groups of doctors, from Oman and regional countries, is under consideration.
Hypothermia prolongs activation of NF-κΒ and augments generation of inflammatory cytokines
Authors: Fairchild Karen D. , Singh Ishwar S. , Patel Sandip , Drysdale Beth E. , Viscardi Rose M. , Hester Lisa , Lazusky Heather M. , Hasday Jeffrey D.
Published: April 2004

While moderate hypothermia is protective against ischemic cardiac and brain injury, it is associated with much higher mortality in patients with sepsis. We previously showed that in vitro exposure to moderate hypothermia (32°C) delays the induction and prolongs the duration of TNF-α and IL-1β secretion by lipopolysaccharide (LPS)-stimulated human mononuclear phagocytes. In the present study, we extended these observations by showing that moderate hypothermia exerts effects on TNF-α and IL-1β generation in the human THP-1 monocyte cell line that are similar to those that we previously found in primary cultured monocytes; that hypothermia causes comparable changes in cytokine generation stimulated by zymosan, toxic shock syndrome toxin-1, and LPS; and that hypothermia causes similar changes in TNF-α and IL-1β mRNA accumulation. TNF-α mRNA half-life, determined after transcriptional arrest with actinomycin D, was not significantly prolonged by lowering incubation temperature from 37 to 32°C, suggesting that hypothermia modifies TNF-α gene transcription. This finding was further supported by reporter gene studies showing a threefold increase in activity of the human TNF-α promoter at 32 vs. 37°C. Electrophoretic mobility shift assay revealed that hypothermia prolonged NF-κΒ activation, identifying a potential role for this transcription factor in mediating the effects of hypothermia on TNF-α and IL-1β production. Delayed reexpression of the inhibitor IκBα, shown by Northern blotting and immunoblotting, may account in part for the prolonged NF-κΒ activation at 32°C. Augmentation of NF-κΒ-dependent gene expression during prolonged exposure to hypothermia may be a common mechanism leading to increased lethality in sepsis, late-onset systemic inflammatory response syndrome after accidental hypothermia, and neuroprotection after ischemia.