bluuebunny/crossref_metadata_2025_split huggingface dataset:
Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1
Authors: Beaudoin Jude , Labbé Simon 
Published: February 2006

ABSTRACT

Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In
Schizosaccharomyces pombe
, the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys
13
, Arg
16
, Arg
19
, Lys
24
, Arg
28
, Lys
45
, Arg
47
, Arg
50
, and Arg
53
residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif,
328
Cys-X-Cys-X
3
-Cys-X-Cys-X
2
-Cys-X
2
-His
342
(designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in
trans
and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.
Ebs1p, a Negative Regulator of Gene Expression Controlled by the Upf Proteins in the Yeast
Saccharomyces cerevisiae
Authors: Ford Amanda S. , Guan Qiaoning , Neeno-Eckwall Eric , Culbertson Michael R. 
Published: February 2006

ABSTRACT

Mutations in
EBS1
were identified in
Saccharomyces cerevisiae
that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of
EBS1
suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of
EBS1
function causes suppression by increasing the rate of translation. Changes in
EBS1
expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of
EBS1
. Translation of a
lacZ
reporter was increased in strains carrying an
ebs1-Δ
mutant gene, whereas translation was decreased when
EBS1
was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the
MFA2
transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally,
EBS1
transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD.
The Function and Properties of the Azf1 Transcriptional Regulator Change with Growth Conditions in
Saccharomyces cerevisiae
Authors: Slattery Matthew G. , Liko Dritan , Heideman Warren 
Published: February 2006

ABSTRACT

Azf1 activates
CLN3
transcription in
Saccharomyces cerevisiae
cells growing in glucose. Paradoxically, other studies have shown Azf1 to be almost undetectable in glucose-grown cells. Microarray experiments showed that Azf1 activates nonoverlapping gene sets in different carbon sources: in glucose, Azf1 activates carbon and energy metabolism genes, and in glycerol-lactate, Azf1 activates genes needed for cell wall maintenance. Consistent with the decreased expression of cell wall maintenance genes observed with
azf1
Δ mutants, we observed a marked growth defect in the
azf1
Δ cells at 37°C in nonfermentable medium. Cell wall integrity assays, such as sensitivity to calcofluor white, sodium dodecyl sulfate, or caffeine, confirmed cell wall defects in
azf1
Δ mutants in nonfermentable medium. Gel shift experiments show that Azf1 binds to DNA elements with the sequence AAAAGAAA (A
4
GA
3
), a motif enriched in the promoters of Azf1-sensitive genes and predicted by whole-genome studies. This suggests that many of the Azf1-dependent transcripts may be regulated directly by Azf1 binding. We found that the levels of Azf1 protein in glucose-grown cells were comparable to Azf1 levels in cells grown in glycerol-lactate; however, this could only be demonstrated with a cell extraction procedure that minimizes proteolysis. Glucose produces conditions that destabilize the Azf1 protein, a finding that may reflect a glucose-induced change in Azf1 tertiary or quaternary structure.
Rac1 and Cdc42 Have Different Roles in
Candida albicans
Development
Authors: Bassilana Martine , Arkowitz Robert A. 
Published: February 2006

ABSTRACT

We investigated the role of the highly conserved G protein Rac1 in the opportunistic pathogen
Candida albicans
. We identified and disrupted
RAC1
and show here that, in contrast to
CDC42
, it is not necessary for viability or serum-induced hyphal growth but is essential for filamentous growth when cells are embedded in a matrix. Rac1 is localized to the plasma membrane, yet its distribution is more homogenous than that of Cdc42, with no enrichment at the tips of either buds or hyphae. In addition, fluorescence recovery after photobleaching results indicate that Rac1 and Cdc42 have different dynamics at the membrane. Furthermore, overexpression of Rac1 does not complement Cdc42 function, and conversely, overexpression of Cdc42 does not complement Rac1 function. Thus, Rac1 and Cdc42, although highly similar to one another, have different roles in
C. albicans
development.
The Cek1 and Hog1 Mitogen-Activated Protein Kinases Play Complementary Roles in Cell Wall Biogenesis and Chlamydospore Formation in the Fungal Pathogen
Candida albicans
Authors: Eisman B. , Alonso-Monge R. , Román E. , Arana D. , Nombela C. , Pla J. 
Published: February 2006

ABSTRACT

The Hog1 mitogen-activated protein (MAP) kinase mediates an adaptive response to both osmotic and oxidative stress in the fungal pathogen
Candida albicans
. This protein also participates in two distinct morphogenetic processes, namely the yeast-to-hypha transition (as a repressor) and chlamydospore formation (as an inducer). We show here that repression of filamentous growth occurs both under serum limitation and under other partially inducing conditions, such as low temperature, low pH, or nitrogen starvation. To understand the relationship of the HOG pathway to other MAP kinase cascades that also play a role in morphological transitions, we have constructed and characterized a set of double mutants in which we deleted both the
HOG1
gene and other signaling elements (the
CST20
,
CLA4
, and
HST7
kinases, the
CPH1
and
EFG1
transcription factors, and the
CPP1
protein phosphatase). We also show that Hog1 prevents the yeast-to-hypha switch independent of all the elements analyzed and that the inability of the
hog1
mutants to form chlamydospores is suppressed when additional elements of the
CEK1
pathway (
CST20
or
HST7
) are altered. Finally, we report that Hog1 represses the activation of the Cek1 MAP kinase under basal conditions and that Cek1 activation correlates with resistance to certain cell wall inhibitors (such as Congo red), demonstrating a role for this pathway in cell wall biogenesis.
Physiological Importance and Identification of Novel Targets for the N-Terminal Acetyltransferase NatB
Authors: Caesar Robert , Warringer Jonas , Blomberg Anders 
Published: February 2006

ABSTRACT

The N-terminal acetyltransferase NatB in
Saccharomyces cerevisiae
consists of the catalytic subunit Nat3p and the associated subunit Mdm20p. We here extend our present knowledge about the physiological role of NatB by a combined proteomics and phenomics approach. We found that strains deleted for either
NAT3
or
MDM20
displayed different growth rates and morphologies in specific stress conditions, demonstrating that the two NatB subunits have partly individual functions. Earlier reported phenotypes of the
nat3Δ
strain have been associated with altered functionality of actin cables. However, we found that point mutants of tropomyosin that suppress the actin cable defect observed in
nat3Δ
only partially restores wild-type growth and morphology, indicating the existence of functionally important acetylations unrelated to actin cable function. Predicted NatB substrates were dramatically overrepresented in a distinct set of biological processes, mainly related to DNA processing and cell cycle progression. Three of these proteins, Cac2p, Pac10p, and Swc7p, were identified as true NatB substrates. To identify N-terminal acetylations potentially important for protein function, we performed a large-scale comparative phenotypic analysis including
nat3Δ
and strains deleted for the putative NatB substrates involved in cell cycle regulation and DNA processing. By this procedure we predicted functional importance of the N-terminal acetylation for 31 proteins.
Multifarious Transcriptional Regulation of Adhesion Protein Gene
ap65
-
1
by a Novel Myb1 Protein in the Protozoan Parasite
Trichomonas vaginalis
Authors: Ong Shiou-Jeng , Hsu Hong-Ming , Liu Hsing-Wei , Chu Chien-Hsin , Tai Jung-Hsiang 
Published: February 2006

ABSTRACT

The transcription efficiency of an adhesion protein gene,
ap65
-
1
, in
Trichomonas vaginalis
varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an
ap65
-
1
reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A
myb1
gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in
T. vaginalis
resulted in repression or activation of
ap65
-
1
transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible
ap65
-
1
transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal
ap65
-
1
promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious
ap65
-
1
transcription, possibly via differential selection of multiple promoter sites upon environmental changes.
The Phosducin-Like Protein PhnA Is Required for Gβγ-Mediated Signaling for Vegetative Growth, Developmental Control, and Toxin Biosynthesis in
Aspergillus nidulans
Authors: Seo Jeong-Ah , Yu Jae-Hyuk 
Published: February 2006

ABSTRACT

Phosducin or phosducin-like protein (PhLP) is a positive regulator of Gβγ activity. The Gβ (SfaD) and Gγ (GpgA) subunits function in vegetative growth and developmental control in the model filamentous fungus
Aspergillus nidulans
. To better understand the nature of Gβγ-mediated signaling,
phnA
, encoding an
A. nidulans
PhLP, has been studied. Deletion of
phnA
resulted in phenotypes almost identical to those caused by deletion of
sfaD
, i.e., reduced biomass, asexual sporulation in liquid submerged culture, and defective fruiting body formation, suggesting that PhnA is necessary for Gβ function. The requirement for the RGS protein FlbA in asexual sporulation could be bypassed by the Δ
phnA
mutation, indicating that PhnA functions in FlbA-controlled vegetative growth signaling, primarily mediated by the heterotrimeric G protein composed of FadA (Gα), SfaD, and GpgA. However, whereas deletion of
fadA
restored both asexual sporulation and the production of sterigmatocystin (ST), deletion of
sfaD
,
gpgA
, or
phnA
failed to restore ST production in the Δ
flbA
mutant. Further studies revealed that SfaD, GpgA, and PhnA are necessary for the expression of
aflR
, encoding the transcriptional activator for the ST biosynthetic genes, and subsequent ST biosynthesis. Overexpression of
aflR
bypassed the need for SfaD in ST production, indicating that the results of SfaD-mediated signaling may include transcriptional activation of
aflR
. Potential differential roles of FadA, Gβγ, and FlbA in controlling ST biosynthesis are further discussed.
Vacuolar Membrane Dynamics in the Filamentous Fungus
Aspergillus oryzae
Authors: Shoji Jun-ya , Arioka Manabu , Kitamoto Katsuhiko 
Published: February 2006

ABSTRACT

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Ao
vam3
, the homologue of the
Saccharomyces cerevisiae VAM3
gene that encodes the vacuolar syntaxin, from
Aspergillus oryzae
. In yeast complementation analyses, the expression of Ao
vam3
restored the phenotypes of both Δ
vam3
and Δ
pep12
mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in
A. oryzae
. FM4-64 [
N
-(3-triethylammoniumpropyl)-4-(
p
-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.
Metallothionein Gene from
Tetrahymena thermophila
with a Copper-Inducible-Repressible Promoter
Authors: Boldrin Francesco , Santovito Gianfranco , Gaertig Jacek , Wloga Dorota , Cassidy-Hanley Donna , Clark Theodore G. , Piccinni Ester 
Published: February 2006

ABSTRACT

We describe a novel metallothionein gene from
Tetrahymena thermophila
that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in
Tetrahymena
and enhancing its applications in cell and molecular biology, as well as biotechnology.
Nuclear β-catenin and Ki-67 expression in choriocarcinoma and its pre-malignant form
Authors: Wong S C C , Chan A T C , Chan J K C , Lo Y M D 
Published: February 2006

Objective:To study the expression of nuclear β-catenin and Ki-67 in patients with normal gestation products (NGP), complete hydatidiform moles (CHM), and choriocarcinoma to elucidate their roles in carcinogenesis and their interrelations.Methods:Expression of nuclear β-catenin and Ki-67 was studied by immunohistochemistry using paraffin embedded blocks. Sixty NGP, 60 CHM, and 10 choriocarcinomas were analysed. In addition, approximately 400 trophoblasts each in 40 NGP, 40 CHM, and 10 choriocarcinomas from the same batch of samples were microdissected for quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) to compareβ-cateninmRNA concentration among them.Results:In the chorionic villi of NGP, β-catenin was consistently expressed in the nuclei of cytotrophoblasts but not syncytiotrophoblasts. Nuclear β-catenin expression was comparatively reduced in CHM trophoblasts and was absent in choriocarcinoma. By contrast, Ki-67 expression was increased from cytotrophoblasts but not in syncytiotrophoblasts in the chorionic villi of NGP to CHM trophoblasts and choriocarcinoma. Using Q-RT-PCR,β-cateninmRNA was detected in 10 NGP, 13 CHM, and three choriocarcinoma specimens, with median copy numbers of 43 230, 18 229, and 17 334 per 400 trophoblasts, respectively. A housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was detected as a control in the NGP, CHM, and choriocarcinoma specimens, with median copy numbers of 51 300, 54 270, and 97 150 per 400 trophoblasts, respectively. Thus medianβ-cateninmRNA values after normalisation were 0.85 in NGP (n = 10), 0.31 in CHM (n = 13), and 0.16 in choriocarcinoma (n = 3).Conclusions:Decreased nuclear β-catenin expression and increased Ki-67 expression may be involved in choriocarcinoma carcinogenesis. The findings also suggest that nuclear β-catenin may play a role in trophoblast differentiation during normal placental development.
Cyclooxygenase-2 expression correlates with poor prognosis in pancreatic cancer
Authors: Juuti A , Louhimo J , Nordling S , Ristimäki A , Haglund C 
Published: February 2006

Background:Cyclooxygenase-2 (COX-2) overexpression is related to poor outcome in several cancers. COX-2 is upregulated in 42–90% of pancreatic ductal adenocarcinomas and is a potential target for chemotherapy. Earlier studies have not shown the expression of COX-2 to be a prognostic factor in pancreatic cancer.Objective:To evaluate the prognostic value of COX-2 in a series of patients with pancreatic adenocarcinoma.Methods:128 patients operated on for pancreatic adenocarcinoma at Helsinki University Central Hospital between 1974 and 1998 provided sections from primary tumours which were immunohistochemically stained with a COX-2-antihuman monoclonal antibody.Results:Cytoplasmic COX-2 reactivity (>5%) occurred in 46 specimens (36%), correlating neither with age, sex, stage, size, tumour stage, nodal metastases, nor grade. Lack of COX-2 expression correlated with distant metastases (p = 0.026). In univariate survival analysis, COX-2 expression (p = 0.0114), stage (p = 0.0002), grade (p = 0.0001), and age (p = 0.042) had prognostic significance. One, two, and five year survival rates were 51%, 32%, and 8% in the COX-2 negative groups compared with 34%, 5%, and 5% in the COX-2 positive groups (p = 0.011). Prognostic significance was especially high for patients operated on with curative intent (p = 0.004). In multivariate analysis, COX-2 was an independent prognostic factor (hazard ratio = 1.6 (95% confidence interval, 1.1 to 2.3)).Conclusions:Expression of COX-2 was associated with poor outcome from pancreatic ductal adenocarcinoma and was independent of tumour stage, grade, or age in multivariate analysis.
Correlation of clinicopathological features with immunohistochemical expression of cell cycle regulatory proteins p16 and retinoblastoma: distinct association with keratinisation and differentiation in oral cavity squamous cell carcinoma
Authors: Muirhead D M , Hoffman H T , Robinson R A 
Published: February 2006

Background:Thep16and retinoblastoma (Rb) gene products are part of the retinoblastoma pathway controlling the G1–S transition of the cell cycle. Few studies on the expression of p16 and retinoblastoma proteins in oral cavity squamous carcinomas have been conducted.Aim:To correlate the expression of p16 and retinoblastoma proteins to clinicopathological characteristics in these tumours.Methods:45 patients with resected oral cavity squamous carcinoma were selected, for whom this was the initial treatment and who were followed up for 5 years or until death. Immunohistochemical stains with antibodies to theRbandp16gene products were carried out on paraffin wax-embedded tissue. Data on clinicopathological features such as tumour differentiation, nodal status, stage and survival outcome were collected.Results:Retinoblastoma expression was seen in 39 of 45 (87%) patients and p16 expression in 6 of 45 (13%) patients. A significant inverse correlation was observed between retinoblastoma and p16 expression as nearly all retinoblastoma negative cases were p16 positive, and vice versa. When examined for clinicopathological correlates, it was found that all 39 tumours that expressed retinoblastoma displayed marked keratinisation and were of low–moderate histological grade. Conversely, five of the six tumours that expressed p16 were found to be poorly differentiated, with minimal keratin expression.Conclusions:Salient relationships were seen between expression of retinoblastoma and p16 and keratinisation. A marked loss of keratin production was evident in the tumours that expressed p16. Tumours expressing retinoblastoma were seen to exhibit more widespread keratinisation. In addition, an inverse staining pattern was found for retinoblastoma and p16 as retinoblastoma-expressing tumours were nearly universally p16 negative and vice versa. No correlation of expression of either p16 or retinoblastoma was found with survival or stage. A link between the histologically observable morphology and expression of cell cycle regulatory protein with the expression of p16 and retinoblastoma has been suggested with keratinisation and differentiation of status.
Metaplastic carcinoma of the breast: a clinicopathological review
Authors: Tse G M , Tan P H , Putti T C , Lui P C W , Chaiwun B , Law B K B 
Published: February 2006

Background:Mammary metaplastic carcinoma encompasses epithelial-only carcinoma (high-grade adenosquamous carcinoma or pure squamous cell carcinoma), biphasic epithelial and sarcomatoid carcinoma and monophasic spindle cell carcinoma.Aim:To evaluate the clinicopathological features of a large series of 34 metaplastic carcinomas.Methods:10 epithelial-only, 14 biphasic and 10 monophasic metaplastic carcinomas were assessed for nuclear grade, hormone receptor status, HER2/neu (cerbB2) oncogene expression, Ki-67 and p53, lymph node status and recurrence on follow-up.Results:Intermediate to high nuclear grade were assessed in most (33/34) tumours. Oestrogen and progesterone receptors were negative in 8 of 10 epithelial-only, all 14 biphasic, and 9 of 10 monophasic tumours, cerbB2 was negative in 7 of 10 epithelial-only, all 14 biphasic and 8 of 10 monophasic tumours. Ki-67 was found to be positive in 6 of 10 epithelial-only, 6 of 14 biphasic, and 7 of 10 monophasic tumours, whereas p53 was positive in 6 of 10 epithelial-only, 7 of 14 biphasic, and 8 of 10 monophasic tumours. Lymph node metastases were seen in 7 of 7 epithelial-only, 7 of 11 biphasic, and 3 of 7 monophasic tumours. Recurrences were seen in 4 of 7 epithelial-only, 8 of 9 biphasic, and 4 of 9 monophasic tumours.Conclusions:All three subtypes of metaplastic carcinoma are known to behave aggressively, and should be differentiated from the low-grade fibromatosis-like metaplastic carcinoma, which does not metastasise. Oncological treatment options may be limited by the frequently negative status of hormonal receptor and cerbB2.