bluuebunny/crossref_metadata_2025 huggingface dataset:
General Anesthesia Delays the Inflammatory Response and Increases Survival for Mice with Endotoxic Shock
Authors: Fuentes Joseph M. , Talamini Mark A. , Fulton William B. , Hanly Eric J. , Aurora Alexander R. , De Maio Antonio
Published: February 2006

Anesthesia is an indispensable component of any operative procedure. In this study, we demonstrate that continuous isoflurane anesthesia for 1 h after a lethal dose (20 mg/kg of body weight) ofEscherichia colilipopolysaccharide (LPS) results in a significant increase in survival of C57BL/6J (B6) mice in comparison with survival of nonanesthetized mice. Protection by anesthesia correlates with a delay in plasma LPS circulation, resulting in a delayed inflammatory response, particularly DNA binding activity of NF-κB and serum levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10. Disparate classes of anesthetic agents produce the same effects on the inflammatory response, which is also independent of the inbred mouse strain used. These results suggest that anesthesia has an important impact on the outcome from endotoxemia. Moreover, the immunomodulatory effects of anesthetics should be considered when interpreting data from experimental animal models.
Anti-Interleukin-15 Prevents Arthritis inBorrelia-Vaccinated and -Infected Mice
Authors: Amlong Corey A. , Nardelli Dean T. , Peterson Sara Heil , Warner Thomas F. , Callister Steven M. , Schell Ronald F.
Published: February 2006

We showed previously that interleukin-17 (IL-17) plays a significant role in the induction of arthritis associated withBorreliavaccination and challenge. Little information, however, is available about the chain of immunologic events that leads to the release of IL-17. The production of IL-17 has been linked to stimulation of memory cells by IL-15. Therefore, we hypothesized that IL-15 is involved in the induction of arthritis associated withBorreliavaccination and infection of mice. Here we present evidence that treatment ofBorrelia-vaccinated and -infected mice with anti-IL-15 antibody prevents swelling of the hind paws. More importantly, both anti-IL-15 antibody- and recombinant IL-15 receptor alpha-treatedBorrelia-vaccinated and -infected mice were free of major histopathologic indications of arthritis, including hyperplasia, hypertrophy, and vilus formation of the synovium. Similarly, the synovial space and perisynovium were free of inflammatory cells. By contrast, the synovium of nontreatedBorrelia-vaccinated and -infected mice had overt hyperplasia, hypertrophy, and vilus formation. Moreover, the synovial space and perisynovium were infiltrated with neutrophils, macrophages, and lymphocytes. Finally, we show that recombinant IL-15 stimulates the release of IL-17 from lymph node cells obtained near the arthritic site. These results suggest that IL-15 plays a major role in orchestrating IL-17 induction of arthritis associated withBorrelia-vaccinated and -infected mice.
Development of Specific Immunoglobulin Ga (IgGa) and IgGb Antibodies Correlates with Control of Parasitemia in
Babesia equi
Infection
Authors: Cunha Cristina W. , McGuire Travis C. , Kappmeyer Lowell S. , Hines Stephen A. , Lopez A. Marianela , Dellagostin Odir A. , Knowles Donald P.
Published: February 2006

In this study, the kinetics of specific immunoglobulin G (IgG) isotypes were characterized in
Babesia equi
(
Theileria equi
)-infected horses. IgGa and IgGb developed during acute infection, whereas IgG(T) was detected only after resolution of acute parasitemia. The same IgG isotype profile induced during acute infection was obtained by equi merozoite antigen 1/saponin immunization.
Differential Expression of
Aspergillus nidulans
Ammonium Permease Genes Is Regulated by GATA Transcription Factor AreA
Authors: Monahan Brendon J. , Askin Marion C. , Hynes Michael J. , Davis Meryl A.
Published: February 2006

The movement of ammonium across biological membranes is mediated in both prokaryotes and eukaryotes by ammonium transport proteins (AMT/MEP) that constitute a family of related sequences. We have previously identified two ammonium permeases in
Aspergillus nidulans
, encoded by the
meaA
and
mepA
genes. Here we show that
meaA
is expressed in the presence of ammonium, consistent with the function of MeaA as the main ammonium transporter required for optimal growth on ammonium as a nitrogen source. In contrast,
mepA
, which encodes a high-affinity ammonium permease, is expressed only under nitrogen-limiting or starvation conditions. We have identified two additional AMT/MEP-like genes in
A. nidulans
, namely,
mepB
, which encodes a second high-affinity ammonium transporter expressed only in response to complete nitrogen starvation, and
mepC
, which is expressed at low levels under all nitrogen conditions. The MepC gene product is more divergent than the other
A. nidulans
AMT/MEP proteins and is not thought to significantly contribute to ammonium uptake under normal conditions. Remarkably, the expression of each AMT/MEP gene under all nitrogen conditions is regulated by the global nitrogen regulatory GATA factor AreA. Therefore, AreA is also active under nitrogen-sufficient conditions, along with its established role as a transcriptional activator in response to nitrogen limitation.
MNN5
Encodes an Iron-Regulated α-1,2-Mannosyltransferase Important for Protein Glycosylation, Cell Wall Integrity, Morphogenesis, and Virulence in
Candida albicans
Authors: Bai Chen , Xu Xiao-Li , Chan Fong-Yee , Lee Raymond Teck Ho , Wang Yue
Published: February 2006

The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus
Candida albicans
. We previously identified a
C. albicans
α-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in
Saccharomyces cerevisiae
. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both α-1,2- and α-1,6-mannobiose, and this activity requires Mn
2+
as a cofactor and is regulated by the Fe
2+
concentration. An
mnn5Δ
mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The
mnn5Δ
mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the
mnn5Δ
mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of
C. albicans
. In summary,
MNN5
deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.
Heterologous Expression of Membrane and Soluble Proteins Derepresses
GCN4
mRNA Translation in the Yeast
Saccharomyces cerevisiae
Authors: Steffensen Lotte , Pedersen Per Amstrup
Published: February 2006

This paper describes the first physiological response at the translational level towards heterologous protein production in
Saccharomyces cerevisiae
. In yeast, the phosphorylation of eukaryotic initiation factor 2α (eIF-2α) by Gcn2p protein kinase mediates derepression of
GCN4
mRNA translation. Gcn4p is a transcription factor initially found to be required for transcriptional induction of genes responsible for amino acid or purine biosynthesis. Using various
GCN4-lacZ
fusions, knockout yeast strains, and anti-eIF-2α-P/anti-eIF-2α antibodies, we observed that heterologous expression of the membrane-bound α
1
β
1
Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase α
1
subunit derepressed
GCN4
mRNA translation up to 70-fold.
GCN4
translation was very sensitive to the presence of heterologous protein, as a density of 1‰ of heterologous membrane protein derepressed translation maximally. Translational derepression of
GCN4
was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased
GCN4
translation to the same extent. In situ activity of the heterologously expressed protein was not required for derepression of
GCN4
mRNA translation, as illustrated by the expression of an enzymatically inactive Na,K-ATPase. Two- to threefold overexpression of the highly abundant and plasma membrane-located endogenous H-ATPase also induced
GCN4
translation. Derepression of
GCN4
translation required phosphorylation of eIF-2α, the tRNA binding domain of Gcn2p, and the ribosome-associated proteins Gcn1p and Gcn20p. The increase in Gcn4p density in response to heterologous expression did not induce transcription from the
HIS4
promoter, a traditional Gcn4p target.
Role of N-Terminal Hydrophobic Region in Modulating the Subcellular Localization and Enzyme Activity of the Bisphosphate Nucleotidase from
Debaryomyces hansenii
Authors: Aggarwal Monika , Mondal Alok K.
Published: February 2006

3′, 5′-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3′-phosphoadenosine-5′-phosphate to adenosine-5′-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (
DHAL2
) from the halotolerant yeast
Debaryomyces hansenii
. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism.
Retention and Loss of Amino Acid Biosynthetic Pathways Based on Analysis of Whole-Genome Sequences
Authors: Payne Samuel H. , Loomis William F.
Published: February 2006

Plants and fungi can synthesize each of the 20 amino acids by using biosynthetic pathways inherited from their bacterial ancestors. However, the ability to synthesize nine amino acids (Phe, Trp, Ile, Leu, Val, Lys, His, Thr, and Met) was lost in a wide variety of eukaryotes that evolved the ability to feed on other organisms. Since the biosynthetic pathways and their respective enzymes are well characterized, orthologs can be recognized in whole genomes to understand when in evolution pathways were lost. The pattern of pathway loss and retention was analyzed in the complete genomes of three early-diverging protist parasites, the amoeba
Dictyostelium
, and six animals. The nine pathways were lost independently in animals,
Dictyostelium
,
Leishmania
,
Plasmodium
, and
Cryptosporidium
. Seven additional pathways appear to have been lost in one or another parasite, demonstrating that they are dispensable in a nutrition-rich environment. Our predictions of pathways retained and pathways lost based on computational analyses of whole genomes are validated by minimal-medium studies with mammals, fish, worms, and
Dictyostelium
. The apparent selective advantages of retaining biosynthetic capabilities for amino acids available in the diet are considered.
Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1
Authors: Beaudoin Jude , Labbé Simon
Published: February 2006

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

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

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

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

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

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

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

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

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

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.
Immunocytochemistry of p16INK4ain liquid-based cervicovaginal specimens with modified Papanicolaou counterstaining
Authors: Negri G , Moretto G , Menia E , Vittadello F , Kasal A , Mian C , Egarter-Vigl E
Published: February 2006

Aim:To evaluate the feasibility and value of a modified Papanicolaou counterstain for p16INK4aimmunostaining in liquid-based cervicovaginal samples.

Methods:Immunocytochemical analyses were carried out with p16INK4aand modified Papanicolaou counterstain on 81 liquid-based samples, including 23 of within normal limits (WNL), 6 of low-grade squamous intraepithelial lesion (LSIL), 20 of high-grade squamous intraepithelial lesion (HSIL), 16 of atypical squamous cells of undetermined significance (ASC-US) and 16 of atypical squamous cells, high-grade lesion cannot be excluded (ASC-H). Results were compared with histological or cytological follow-up. For comparison, samples from 29 more cases (10 of LSIL, 10 of ASC-H and 9 of HSIL) were immunostained with p16INK4aand conventionally counterstained with haematoxylin. The intensity of immunostaining in cases of squamous intraepithelial lesion (SIL) was assessed using a 0–3 scoring system. Interobserver agreement was calculated by κ statistics.

Results:Expression of p16INK4awas detected in 3 of 23 cases of WNL, 4 of 6 cases of LSIL, all cases of HSIL, 5 of 16 cases of ASC-US and 13 of 16 cases of ASC-H. Excluding two cases with no residual dysplastic cells in the immunocytochemistry, all cases of cervical intraepithelial neoplasia (CIN)2 or CIN3 at follow-up expressed p16INK4aand none of the p16INK4a-negative cases showed a high-grade lesion at follow-up. No evident differences in pattern or intensity of p16INK4aexpression were observed between the specimens of the study and control groups. Interobserver agreement was significantly better in the study group than in the group with conventional immunostaining (combined κ 0.773v0.549; p<0.05), and still better, albeit statistically not significant, than with conventional immunostaining and cervical smear test together (combined κ 0.773v0.642).

Conclusion:Immunocytochemistry with p16INK4aand modified Papanicolaou counterstain may add to the cervicovaginal cytology the full potentiality of p16INK4awithout the need of a further slide and the risk of loss of dysplastic cells, yet maintaining the typical morphological features of the smear test.
Oncocytic change in pleomorphic adenoma: molecular evidence in support of an origin in neoplastic cells
Authors: Di Palma Silvana , Lambros Maryou B K , Savage Kay , Jones Chris , Mackay Alan , Dexter Tim , Iravani Marjan , Fenwick Kerry , Ashworth Alan , Reis-Filho Jorge S
Published: February 2006

Background:Cells with oncocytic change (OC) are a common finding in salivary glands (SGs) and in SG tumours. When found within pleomorphic adenomas (PAs), cells with OC may be perceived as evidence of malignancy, and lead to a misdiagnosis of carcinoma ex pleomorphic adenoma (CaExPa).

Aim:To describe a case of PA with atypical OC, resembling a CaExPa. A genomewide molecular analysis was carried out to compare the molecular genetic features of the two components and to determine whether the oncocytic cells originated from PA cells, entrapped normal cells, or whether these cells constitute an independent tumour.

Materials and methods:Representative blocks were immunohistochemically analysed with antibodies raised against cytokeratin (Ck) 5/6, Ck8/18, Ck14, vimentin, p63, α-smooth muscle actin (ASMA), S100 protein, anti-mitochondria antibody, β-catenin, HER2, Ki67, p53 and epidermal growth factor receptor. Typical areas of PA and OC were microdissected and subjected to microarray-based comparative genomic hybridisation (aCGH). Chromogenic in situ hybridisation (CISH) was performed with in-house generated probes to validate the aCGH findings.

Results:PA cells showed the typical immunohistochemical profile, including positivity for Ck5/6, Ck8/18, Ck14, vimentin, ASMA, S100 protein, p63, epidermal growth factor receptor and β-catenin, whereas oncocytic cells showed a luminal phenotype, expression of anti-mitochondria antibody and reduced β-catenin staining. Both components showed low proliferation rates and lacked p53 reactivity. aCGH revealed a similar amplification in both components, mapping to 12q13.3–q21.1, which was further validated by CISH. No HER2 gene amplification or overexpression was observed. The foci of oncocytic metaplasia showed an additional low-level gain of 6p25.2–p21.31.

Conclusion:The present data demonstrate that the bizarre atypical cells of the present case show evidence of clonality but no features of malignancy. In addition, owing to the presence of a similar genome amplification pattern in both components, it is proposed that at least in some cases, OC may originate from PA cells.
Bacteraemia caused byAnaerotruncus colihominisand emended description of the species
Authors: Lau S K P , Woo P C Y , Woo G K S , Fung A M Y , Ngan A H Y , Song Y , Liu C , Summanen P , Finegold S M , Yuen K
Published: February 2006

Background:Anaerotruncus colihomonisis a newly described bacterial genus and species isolated from the stool specimens of children. Its clinical significance, however, is unknown.

Aims:To describe a case ofA colihominisbacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing and provide an emended description of the species.

Methods:An unidentified anaerobic bacillus (strain HKU19) that stains Gram negative was subjected to characterisation by 16S rRNA gene sequencing, G+C content determination and electron microscopy.

Results:Strain HKU19 was isolated from the blood culture of a 78-year-old woman with nosocomial bacteraemia. It was found to be an anaerobic, non-motile, pleomorphic, thin bacillus that stains Gram negative. It produces Indole and utilises glucose and mannose. Identifying the strain to the species level was not possible by conventional phenotypic tests and commercial identification systems. The G+C content of strain HKU19 was found to be 53.43 mol%. A similarity of 99.3% nucleotide identities was found between the 16S rRNA gene sequence of strain HKU19 and that ofA colihominisWAL 14 565T, which was isolated from a human faecal specimen. In contrast with the original description ofA colihominis, HKU19 was found to produce occasional oval, terminal spores, although the other phenotypic characteristics matched. Spores were also occasionally observed when the two previously reported strains were re-examined.

Conclusions:Although the source of the bacteraemia in the patient cannot be determined, this report suggests thatA colihominisis of clinical significance. Spore formation is proposed as an emended description ofA colihominis.
Post-traumatic fibro-osseous lesion of the ribs: a relatively under-recognised entity
Authors: Aubert S , Kerdraon O , Conti M , Buob D , Petit S , Leroy X
Published: February 2006

Aim:To report the description of a rare benign osseous lesion affecting the ribs entitled post-traumatic fibro-osseous lesion (PTFOL).

Methods:Seven cases of PTFOLs were retrieved from the archives of the University Hospital of Lille. Histological slides were reviewed and lesions were classified according to the histological patterns described by McDermottet al. Clinical and follow-up data were obtained from the patients’ charts.

Results:PTFOLs occurred principally in men (mean age 31.8 years) with a known or suspected previous chest injury for four of them. No previous cancer was noted. Radiologically, PTFOLs readily presented as an isolated expansive lucency with a sclerotic rim located on the last five ribs. A constant increased uptake of radionucleotide was noted on bone scan. Microscopically, two fibro-osseous, four xanthomatous and one mixed pattern were individualised. Lesions were characterised by a network of anastomosing bone trabeculae without osteoblast lining within a fibrous stroma. A zonal maturation from woven to peripheral lamellar bone was characterised. Central sheets of lipid-laden histiocytes were conspicuous in the xanthomatous type. For each patient, clinical follow-up was excellent, without any recurrence.

Conclusions:This is the second largest series of PTFOLs, which is considered to be a dysplastic healing process after trauma. It may be symptomatic or shown by imaging studies realised for unrelated reason. PTFOL is regularly misdiagnosed with other more common lesions of the ribs, such as fibro-osseous dysplasia, osteoma osteoid and benign fibrous histiocytoma. Its recognition is of importance because no follow-up is needed after resection.
AN EVALUATION OF THE CROSSING NUMBER ON RIBBON 2-KNOTS
Authors: YASUDA TOMOYUKI
Published: February 2006

For an arbitrary 1-knot k1, the spun 2-knot of k1, denoted by spun (k1), is a ribbon 2-knot in R4. Hence for a ribbon 2-knot K2, we can also induce a notion corresponding to the crossing number on a 1-knot, and it is said to be the crossing number of K2, denoted by cr (K2). In this note, we will show that the Alexander polynomial plays an important role in determining the crossing number of a ribbon 2-knot. Lastly, we will prove the following: If k1is a (p,q)-torus knot, then cr ( spun (k1)) is equal to (p - 1)(q - 1).
COMPLETELY DISTINGUISHABLE PROJECTIONS OF SPATIAL GRAPHS
Authors: NIKKUNI RYO
Published: February 2006

A generic immersion of a finite graph into the 2-space with p double points is said to be completely distinguishable if any two of the 2p embeddings of the graph into the 3-space obtained from the immersion by giving over/under information to each double point are not ambient isotopic in the 3-space. We show that only non-trivializable graphs and non-planar graphs have a non-trivial completely distinguishable immersion. We give examples of non-trivial completely distinguishable immersions of several non-trivializable graphs, the complete graph on n vertices and the complete bipartite graph on m + n vertices.
AN EXTENSION OF THE JONES POLYNOMIAL OF CLASSICAL KNOTS
Authors: SHTYLLA BLERTA , ZULLI LOUIS
Published: February 2006

We define a linear algebraic extension of the Jones polynomial of classical knots, and prove that certain key properties of the classical Jones polynomial are properties of the extension. This shows that these properties are linear algebraic in nature, not topological. We identify a topological property of the classical Jones polynomial, that is, a property of the classical Jones polynomial that the extension does not possess. We discuss ortho-projection matrices, ortho-projection graphs, and their Jones polynomials. We classify, up to isomorphism, the connected ortho-projection graphs with at most eight vertices, and show that each such isomorphism class corresponds to a prime alternating classical knot diagram. We give an example of a connected ortho-projection graph with nine vertices that does not correspond to such a diagram.
THE MAPPING CLASS GROUP OF A DISK WITH INFINITELY MANY HOLES
Authors: FABEL PAUL
Published: February 2006

A left orderable completely metrizable topological group is exhibited containing Artin's braid group on infinitely many strands. The group is the mapping class group (rel boundary) of the closed unit disk with a sequence of interior punctures converging to the boundary. This resolves an issue suggested by work of Dehornoy.
2-IRREDUCIBILITY OF SPATIAL GRAPHS
Authors: LEI FENGCHUN , TANIYAMA KOUKI , ZHANG GENGYU
Published: February 2006

An embedded graph G in the 3-sphere S3 is called 2-irreducible if there are no separating spheres, cutting spheres, singular separating spheres, singular cutting spheres or 2-cutting spheres of G. Let D be a 2-disk in S3 that is very good for G. Let G′ be an embedded graph in S3 obtained from G by contracting D to a point. We show that if G′ is 2-irreducible then G is 2-irreducible. By this criterion certain graphs are easily shown to be 2-irreducible. As an application we show a pair of embedded graphs in the 3-sphere which is distinguished by 2-irreducibility.
INTERSECTION GRAPHS FOR STRING LINKS
Authors: MELLOR BLAKE
Published: February 2006

We extend the notion of intersection graphs for chord diagrams in the theory of finite type knot invariants to chord diagrams for string links. We use our definition to develop weight systems for string links via the adjacency matrix of the intersection graphs, and show that these weight systems are related to the weight systems induced by the Conway and Homfly polynomials.