bluuebunny/crossref_metadata_2025 huggingface dataset:
RADICAL CYSTECTOMY FOR PRIMARY BLADDER MALIGNANCY: A 10 YEAR REVIEW
Authors: Louie‐Johnsun Mark W. , Braslis Karl G. , Murphy Donald L. , Neerhut Gregory J. , Grills Richard J.
Published: March 2007

Background:  Radical cystectomy is universally accepted as the definitive treatment for muscle invasive bladder cancer and preventing stage progression in high‐risk groups. There are few recent reviews outside of major international centres. We compared our institution’s results with published literature.

Methods:  Records of 50 consecutive patients treated with radical cystectomy for biopsy‐proven bladder carcinoma between 1995 and 2005 were reviewed.

Results:  The mean age was 70 years and 76% were male. Median follow up was 38 months (1–111 months). Twenty‐three patients (46%) had known history of superficial transitional cell carcinoma and 12 patients (24%) had undergone previously intravesical therapy. Transitional cell carcinomas accounted for 94% of cases and most (76%) were poorly differentiated. Twenty‐four (48%) had disease at pT3 stage or higher. Regional lymph nodes were involved in 35%. Twenty‐three patients (46%) developed recurrence and over half (12 patients) recurred within 12 months. Both lymph node involvement and recurrence were associated with higher pT stage (P < 0.001). All patients with recurrent disease were dead within a year (median 103 days). Median hospital stay was 19 days and there was one postoperative death (2%). Five‐year disease‐free and overall survival were 42 and 34% respectively.

Conclusion:  Survival following curative resection for primary bladder malignancy is at best modest. Our overall proportion of higher stage disease contributed to earlier recurrence and lower survival rates. Our postoperative morbidity and mortality rates as well as length of stay are acceptable compared with major international units.
Biomass‐dependent susceptibility to drought in experimental grassland communities
Authors: Wang Yongfan , Yu Shixiao , Wang Jiang
Published: March 2007

Earlier studies indicated that plant diversity influences community resistance in biomass when ecosystems are exposed to perturbations. This relationship remains controversial, however. Here we constructed grassland communities to test the relationships between species diversity and productivity under control and experimental drought conditions. Species richness was not correlated with biomass either under constant conditions or under drought conditions. However, communities with lower biomass production were more resistant to drought stress than those that were more productive. Our results also showed that ecosystem resistance to drought is a decreasing but nonlinear function of biomass. In contrast, species diversity had little and an equivocal effect on ecosystem resistance. From the results reported here, and the results of several previous studies, we suggest that high biomass systems exhibited a greater biomass reduction in response to drought than low biomass systems did, regardless of the relationship between plant diversity and community biomass production.
Growth‐mortality tradeoffs and ‘personality traits’ in animals
Authors: Stamps Judy A.
Published: March 2007

Consistent individual differences in boldness, reactivity, aggressiveness, and other ‘personality traits’ in animals are stable within individuals but vary across individuals, for reasons which are currently obscure. Here, I suggest that consistent individual differences in growth rates encourage consistent individual differences in behavior patterns that contribute to growth‐mortality tradeoffs. This hypothesis predicts that behavior patterns that increase both growth and mortality rates (e.g. foraging under predation risk, aggressive defense of feeding territories) will be positively correlated with one another across individuals, that selection for high growth rates will increase mean levels of potentially risky behavior across populations, and that within populations, faster‐growing individuals will take more risks in foraging contexts than slower‐growing individuals. Tentative empirical support for these predictions suggests that a growth‐mortality perspective may help explain some of the consistent individual differences in behavioral traits that have been reported in fish, amphibians, reptiles, and other animals with indeterminate growth.
Caribbean mangroves adjust to rising sea level through biotic controls on change in soil elevation
Authors: McKee Karen L. , Cahoon Donald R. , Feller Ilka C.
Published: March 2007

Aim  The long‐term stability of coastal ecosystems such as mangroves and salt marshes depends upon the maintenance of soil elevations within the intertidal habitat as sea level changes. We examined the rates and processes of peat formation by mangroves of the Caribbean Region to better understand biological controls on habitat stability.

Location  Mangrove‐dominated islands on the Caribbean coasts of Belize, Honduras and Panama were selected as study sites.

Methods  Biological processes controlling mangrove peat formation were manipulated (in Belize) by the addition of nutrients (nitrogen or phosphorus) to Rhizophora mangle (red mangrove), and the effects on the dynamics of soil elevation were determined over a 3‐year period using rod surface elevation tables (RSET) and marker horizons. Peat composition and geological accretion rates were determined at all sites using radiocarbon‐dated cores.

Results  The addition of nutrients to mangroves caused significant changes in rates of mangrove root accumulation, which influenced both the rate and direction of change in elevation. Areas with low root input lost elevation and those with high rates gained elevation. These findings were consistent with peat analyses at multiple Caribbean sites showing that deposits (up to 10 m in depth) were composed primarily of mangrove root matter. Comparison of radiocarbon‐dated cores at the study sites with a sea‐level curve for the western Atlantic indicated a tight coupling between peat building in Caribbean mangroves and sea‐level rise over the Holocene.

Main conclusions  Mangroves common to the Caribbean region have adjusted to changing sea level mainly through subsurface accumulation of refractory mangrove roots. Without root and other organic inputs, submergence of these tidal forests is inevitable due to peat decomposition, physical compaction and eustatic sea‐level rise. These findings have relevance for predicting the effects of sea‐level rise and biophysical processes on tropical mangrove ecosystems.
Government and the Reverse‐Holdup Problem
Authors: WICKELGREN ABRAHAM L.
Published: March 2007

When the government bargains with a private firm, the firm cares about only its own profits, but the firm's profits may also enter into the government's utility function. As a result, the government will not bargain as aggressively for a low price. This can lead the government to “over pay” for quality. In contrast to the standard holdup problem, this reverse‐holdup problem can give the firm an incentive to overinvest in non‐contractible quality. The paper also discusses some examples where the reverse‐holdup problem may explain excessive quality in government procurement.
Collective Risk Control and Group Security: The Unexpected Consequences of Differential Risk Aversion
Authors: IHORI TOSHIHIRO , McGUIRE MARTIN C.
Published: March 2007

We provide an analysis of odds‐improving self‐protection for when it yields collective benefits to groups, such as alliances of nations, for whom risks of loss are public bads and prevention of loss is a public good. Our analysis of common risk reduction shows how diminishing returns in risk improvement can be folded into income effects. These income effects then imply that whether protection is inferior or normal depends on the risk aversion characteristics of underlying utility functions, and on the interaction between these, the level of risk, and marginal effectiveness of risk abatement. We demonstrate how public good inferiority is highly likely when the good is “group risk reduction.” In fact, we discover a natural or endogenous limit on the size of a group and of the amount of risk controlling outlay it will provide under Nash behavior. We call this limit an “Inferior Goods Barrier” to voluntary risk reduction. For the paradigm case of declining risk aversion, increases in group size(wealth) will cause provision of more safety to change from a normal to an inferior good thereby creating such a barrier.
P2Y2‐ and P2Y4purinergic receptors are over‐expressed in human colon cancer
Authors: Nylund G. , Hultman L. , Nordgren S. , Delbro D. S.
Published: March 2007

1A characteristic of cancer is altered signal transduction leading to uninhibited growth. Adenosine‐5′‐triphosphate (ATP), a natural ligand at P2X‐ and P2Y purinergic receptors may regulate cell growth in non‐neoplastic, as well as neoplastic tissues. In the human colon cancer cell line, HT‐29, we previously demonstrated the expression of purinergic receptors of the P2Y2‐ and P2Y4subclasses.

2The aim of the current study was to investigate whether these two purinergic receptors are expressed also in human colon cancer, and, if so, how such expression is related to that in tumour‐free colonic tissue.

3The immunohistochemical findings of both P2Y2‐ and P2Y4receptors in the tumours from three patients, prompted us to conduct an investigation of a consecutive series of patients utilizing Western blotting for protein detection and densitometry for quantitation.

4Both P2Y2‐ and P2Y4purinergic receptors could be identified in tumour‐free tissue, and both were significantly over‐expressed in each of the 10 colon cancers.
Long‐wave UVA offers partial protection against UVB‐induced immune suppression in human skin
Authors: SKOV LONE , VILLADSEN LOUISE , ERSBØLL BJARNE K. , SIMON JAN C. , BARKER JONATHAN N. W. N. , BAADSGAARD OLE
Published: March 2007

Ultraviolet‐B (UVB, 280–320 nm) interferes with the generation of cell‐mediated immunity to contact allergens applied epicutaneously on the irradiated site. To investigate whether pretreatment with UVA‐1 (340–400 nm) protects against the UVB‐induced immune suppression we sensitized human volunteers with diphenylcyclopropenone (DPCP) on normal buttock skin (n=12), on UVB‐irradiated buttock skin (n = 21), on buttock skin pretreated with UVA‐1 (n= 12), and on buttock skin pretreated with UVA‐1 and thereafter irradiated with UVB (n=22). Sensitization on UVB‐irradiated skin reduced the immunization rate to DPCP compared with sensitization on non‐irradiated skin (p<0.01) and skin pretreated with UVA‐1 (p<0.01). In contrast, the immunization rate in the group of volunteers sensitized on skin pretreated with UVA‐1 before UVB irradiation was significantly higher than the immunization rate in the group of volunteers sensitized on UVB‐irradiated skin alone (p<0.05). These results indicate that pretreatment with UVA‐1 under certain conditions offers partial protection against the UVB‐induced reduction in the immunization rates to epicutaneous allergens.
Carcinogenicity of estrogens in human breast epithelial cells1
Authors: RUSSO JOSE , HU YUN FU , TAHIN QUIVO , MIHAILA DANA , SLATER CAROLYN , LAREEF M. HASAN , RUSSO IRMA H.
Published: March 2007

Epidemiological and clinical evidences indicate that breast cancer risk is associated with prolonged ovarian function that results in elevated circulating levels of steroid hormones. Principal among these is estrogen, which is associated with two important risk factors, early onset of menarche and late menopause. However, up to now there is no direct experimental evidence that estrogens are responsible of the initiation of human breast cancer. We postulate that if estrogens are causative agents of this disease, they should elicit in human breast epithelial cells (HBEC) genomic alterations similar to those exhibited by human breast cancers, such as DNA amplification and loss of genetic material representing tumor suppressor genes. These effects could result from binding of the hormone to its nuclear receptors (ER) or from its metabolic activation to reactive metabolites. This hypothesis was tested by treating with the natural estrogen 17β‐estradiol (E2) and the synthetic steroid diethylstilbestrol (DES) MCF‐10F cells, a HBEC line that is negative for ER. Cells treated with the chemical carcinogen benzo (a) pyrene (BP) served as a positive control of cell transformation. BP‐, E2‐, and DES‐treated MCF‐10F cells showed increases in survival efficiency and colony efficiency in agar methocel, and loss of ductulogenic capacity in collagen gel. The largest colonies were formed by BP‐treated cells, becoming progressively smaller in DES‐ and E2‐treated cells. The loss of ductulogenic capacity was maximal in BP‐, and less prominent in E2‐ and DES‐treated cells. Genomic analysis revealed that E2‐ and DES‐treated cells exhibited loss of heterozygosity in chromosomes 3 and 11, at 3p21, 3p21–21.2, 3p21.1–14.2, and 3p14.2–14.1, and at 11q23.3 and 11q23.1–25 regions, respectively. It is noteworthy that these loci are also affected in breast lesions, such as ductal hyperplasia, carcinoma in situ, and invasive carcinoma. Our data are the first ones to demonstrate that estrogens induce in HBEC phenotypic changes indicative of cell transformation and that those changes are associated with significant genomic alterations that might unravel new pathways in the initiation of breast cancer.
Sucrose fatty acid esters suppress pancreatic secretion accompanied by peptide YY release in pancreatico‐biliary diverted rats
Authors: Hira Tohru , Takahashi Katsuhiko , Hara Hiroshi
Published: March 2007

Our previous study demonstrated that intestinal administration of triglycerides suppressed protein‐induced increases in pancreatic exocrine secretion in pancreatico‐biliary diverted (PBD) rats, though the mechanism has not been clarified. The present study was conducted to determine whether esterified fatty acids or free fatty acids are responsible for this suppression, and whether an esterified fatty acid stimulates secretion of a pancreatic inhibitory hormone, peptide YY (PYY). We examined the effects of cocoa butter or non‐digestible sucrose fatty acid esters on protein‐induced pancreatic secretion in conscious PBD rats whose bile–pancreatic juice was diverted from the proximal small intestine through a catheter. Intraduodenal administration of the protein guanidinated casein hydrolysate (HGC, 150 mg in 1 ml water) enhanced pancreatic protein and trypsin secretion. However, administration of HGC with cocoa butter (100 mg ml−1) partly suppressed the increase in pancreatic secretion, and HGC with a highly esterified sucrose fatty acid ester, F‐10 (100 mg ml−1), completely suppressed it. The low‐esterified, water‐soluble sucrose fatty acid ester F‐160 also completely inhibited protein‐induced pancreatic secretion in the presence or absence of the lipase inhibitor orlistat.In anaesthetized PBD rats, intraduodenal administration of HGC with sucrose ester F‐160 suppressed HGC‐induced pancreatic secretion, and induced PYY secretion more strongly than HGC with sucrose. These results suggest that the esterified fatty acid itself stimulates PYY release in the distal intestine, thereby inhibiting protein‐induced pancreatic secretion.
Early morning impairment in cerebral autoregulation and cerebrovascular CO2 reactivity in healthy humans: relation to endothelial function
Authors: Ainslie Philip N. , Murrell Carissa , Peebles Karen , Swart Marianne , Skinner Margot A. , Williams Michael J. A. , Taylor Robin D.
Published: March 2007

The reduction in cerebrovascular reactivity to CO2 and/or endothelial function that occurs in the early hours after waking are potential causes for the increased risk for cardiovascular events at this time point. It is unknown whether cerebral autoregulation is reduced in the morning. We tested the hypothesis that early morning reduction in endothelium‐dependent vascular reactivity would be linked to changes in cerebrovascular reactivity to CO2 and cerebral autoregulation (CA). Overnight changes in a dynamic cerebral autoregulation index (ARI) were determined from continuous recordings of blood flow velocity in the middle cerebral artery (MCAv) and arterial blood pressure (BP) during transiently induced hypotension in 20 individuals. Frontal cortical oxygenation (near infrared spectroscopy) and cerebral haemodynamics were also monitored during hypercapnia and before and during 3 min of active standing. Brachial artery flow‐mediated endothelium‐dependent vasodilatation (FMD) and endothelium‐independent dilatation (NFMD) were also monitored. From evening to morning, there was a significant lowering in ARI (5.3 ± 0.5 versus 4.7 ± 0.6 a.u.; P < 0.05), cerebrovascular reactivity to CO2 (5.3 ± 0.6 versus 4.6 ± 1.1% mmHg−1; P < 0.05) and FMD (7.6 ± 0.9 versus 6.0 ± 1.4%; P < 0.05). The lowered FMD was related to the decrease in cerebrovascular reactivity to CO2 (r= 0.76; P < 0.05). Transient reductions in morning MCAv and cortical oxyhaemoglobin concentrations were observed upon resuming a supine‐to‐upright position (P < 0.05 versus evening). The early morning reduction in cerebral autoregulation may facilitate the onset of cerebrovascular accidents; this may be of particular relevance to at‐risk groups, especially upon resuming the upright position.
Effect of Exogenous Reductant on Growth and Iron Mobilization from Ferrihydrite by the
Pseudomonas mendocina ymp
Strain
Authors: Dhungana Suraj , Anthony Charles R. , Hersman Larry E.
Published: March 2007

Growth of the
Pseudomonas mendocina ymp
strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.
Tight Modulation of
Escherichia coli
Bacterial Biofilm Formation through Controlled Expression of Adhesion Factors
Authors: Da Re Sandra , Le Quéré Benjamin , Ghigo Jean-Marc , Beloin Christophe
Published: March 2007

Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development.
Escherichia coli
is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of
E. coli
by taking control of two biofilm-promoting factors, autotransported antigen 43 adhesin and the
bscABZC
cellulose operon. The modulation of the biofilm formation capacities of
E. coli
or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.
Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry
Authors: Berney Michael , Hammes Frederik , Bosshard Franziska , Weilenmann Hans-Ulrich , Egli Thomas
Published: March 2007

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of
Escherichia coli
,
Salmonella enterica
serovar Typhimurium,
Shigella flexneri
, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium
E. coli
as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive
Enterococcus faecalis
, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.
Irradiation Sensitivity of Planktonic and Biofilm-Associated
Escherichia coli
O157:H7 Isolates Is Influenced by Culture Conditions
Authors: Niemira Brendan A.
Published: March 2007

Ionizing radiation effectively inactivates
Escherichia coli
O157:H7, but the efficacy of the process against biofilm cells versus that against free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm cells was determined for three isolates of
E. coli
O157:H7 (C9490, ATCC 35150, and ATCC 43894). Biofilms were formed on sterile glass slides incubated at 37°C for either 24 h, 48 h, or 72 h. The biofilm and planktonic cultures were gamma irradiated at doses ranging from 0.0 (control) to 1.5 kGy. The dose of radiation value required to reduce the population by 90% (D
10
) was calculated for each isolate, culture, and maturity based on viable populations at each radiation dose. For each of the times sampled, the D
10
values of isolate 43894 planktonic cells (0.454 to 0.479 kGy) were significantly (
P
< 0.05) higher than those observed for biofilm cells (0.381 to 0.385 kGy), indicating a significantly increased sensitivity to irradiation for cells in the biofilm habitat. At the 24-h sampling time, isolate C9490 showed a similar pattern, in which the D
10
values of planktonic cells (0.653 kGy) were significantly higher than those for biofilm cells (0.479 kGy), while isolate 35150 showed the reverse, with D
10
values of planktonic cells (0.396 kGy) significantly lower than those for biofilm cells (0.526 kGy). At the 48-h and 72-h sampling times, there were no differences in radiation sensitivities based on biofilm habitat for C9490 or 35150. Biofilm-associated cells, therefore, show a response to irradiation which can differ from that of planktonic counterparts, depending on the isolate and the culture maturity. Culture maturity had a more significant influence on the irradiation efficacy of planktonic cells but not on biofilm-associated cells of
E. coli
O157:H7.
High-Frequency Phage-Mediated Gene Transfer among
Escherichia coli
Cells, Determined at the Single-Cell Level
Authors: Kenzaka Takehiko , Tani Katsuji , Sakotani Akiko , Yamaguchi Nobuyasu , Nasu Masao
Published: March 2007

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among
Escherichia coli
cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated
E. coli
phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10
−8
to 2 × 10
−6
, 1 × 10
−8
to 4 × 10
−8
, and <4 × 10
−9
to 4 × 10
−8
per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10
−4
to 1 × 10
−3
, 9 × 10
−4
to 3 × 10
−3
, and 5 × 10
−4
to 4 × 10
−3
for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.
S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent
Authors: Nomellini John F. , Duncan Gillian , Dorocicz Irene R. , Smit John
Published: March 2007

The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative,Staphylococcus aureusprotein A.Caulobacter crescentusproduces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell asS. aureuscells (Pansorbin). The ability of recombinant protein G-Caulobactercells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobactercells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.
Increased Production of Xylanase by Expression of a Truncated Version of the
xyn11A
Gene from
Nonomuraea flexuosa
in
Trichoderma reesei
Authors: Paloheimo Marja , Mäntylä Arja , Kallio Jarno , Puranen Terhi , Suominen Pirkko
Published: March 2007

We have previously shown that the
Nonomuraea flexuosa
Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the
N. flexuosa xyn11A
gene and the truncated gene was expressed in
Trichoderma reesei
. The xylanase sequence was fused to the
T. reesei
mannanase I (Man5A) signal sequence or 3′ to a
T. reesei
carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (
cel7A
,
cbh1
) promoter. Single-copy isogenic transformants in which the expression cassette replaced the
cel7A
gene were cultivated and analyzed. The transformants expressing the truncated
N. flexuosa xyn11A
produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length
N. flexuosa xyn11A
. The transformant expressing the
cel6A
CBD-truncated
N. flexuosa xyn11A
produced about 1.9 g liter
−1
of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length
N. flexuosa xyn11A
with an exact fusion to the
cel7A
terminator was expressed. We suggest that the
T. reesei
folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length
N. flexuosa xyn11A
is efficiently translated.
Crm1-Mediated Nuclear Export of the
Schizosaccharomyces pombe
Transcription Factor Cuf1 during a Shift from Low to High Copper Concentrations
Authors: Beaudoin Jude , Labbé Simon
Published: March 2007

In this study, we examine the fate of the nuclear pool of the
Schizosaccharomyces pombe
transcription factor Cuf1 in response to variations in copper levels. A nuclear pool of Cuf1-green fluorescent protein (GFP) was generated by expressing a functional
cuf1

+

-
GFP
allele in the presence of a copper chelator. We then extinguished
cuf1

+

-
GFP
expression and tracked the changes in the localization of the nuclear pool of Cuf1-GFP in the presence of low or high copper concentrations. Treating cells with copper as well as silver ions resulted in the nuclear export of Cuf1. We identified a leucine-rich nuclear export signal (NES),
349
LAALNHISAL
358
, within the C-terminal region of Cuf1. Mutations in this sequence abrogated Cuf1 export from the nucleus. Furthermore, amino acid substitutions that impair Cuf1 NES function resulted in increased target gene expression and a concomitant cellular hypersensitivity to copper. Export of the wild-type Cuf1 protein was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export protein Crm1. We further show that cells expressing a temperature-sensitive mutation in
crm1

+

exhibit increased nuclear accumulation of Cuf1 at the nonpermissive temperature. Although wild-type Cuf1 is localized in the nucleus in both conditions, we observed that the protein can still be inactivated by copper, resulting in the repression of
ctr4

+

gene expression in the presence of exogenous copper. These results demonstrate that nuclear accumulation of Cuf1 per se is not sufficient to cause the unregulated expression of the copper transport genes like
ctr4

+

. In addition to nuclear localization, a functional Cys-rich domain or NES element in Cuf1 is required to appropriately regulate copper transport gene expression in response to changes in intracellular copper concentration.
DNA Sequence Characterization and Molecular Evolution of
MAT1
and
MAT2
Mating-Type Loci of the Self-Compatible Ascomycete Mold
Neosartorya fischeri
Authors: Rydholm C. , Dyer P. S. , Lutzoni F.
Published: March 2007

Degenerate PCR and chromosome-walking approaches were used to identify mating-type (
MAT
) genes and flanking regions from the homothallic (sexually self-fertile) euascomycete fungus
Neosartorya fischeri
, a close relative of the opportunistic human pathogen
Aspergillus fumigatus
. Both putative alpha- and high-mobility-group-domain
MAT
genes were found within the same genome, providing a functional explanation for self-fertility. However, unlike those in many homothallic euascomycetes (Pezizomycotina), the genes were not found adjacent to each other and were termed
MAT1
and
MAT2
to recognize the presence of distinct loci. Complete copies of putative
APN1
(DNA lyase) and
SLA2
(cytoskeleton assembly control) genes were found bordering the
MAT1
locus. Partial copies of
APN1
and
SLA2
were also found bordering the
MAT2
locus, but these copies bore the genetic hallmarks of pseudogenes. Genome comparisons revealed synteny over at least 23,300 bp between the
N. fischeri MAT1
region and the
A. fumigatus MAT
locus region, but no such long-range conservation in the
N. fischeri MAT2
region was evident. The sequence upstream of
MAT2
contained numerous candidate transposase genes. These results demonstrate a novel means involving the segmental translocation of a chromosomal region by which the ability to undergo self-fertilization may be acquired. The results are also discussed in relation to their significance in indicating that heterothallism may be ancestral within the
Aspergillus
section
Fumigati
.
l-Arginine and Cationic Amino Acid Transporter 2B Regulate Growth and Survival ofLeishmania amazonensisAmastigotes in Macrophages
Authors: Wanasen Nanchaya , MacLeod Carol L. , Ellies Lesley G. , Soong Lynn
Published: March 2007

Leishmaniaspp. are obligate intracellular parasites, requiring a suitable microenvironment for their growth within host cells. We previously reported that the growth ofLeishmania amazonensisamastigotes in murine macrophages (Mφs) was enhanced in the presence of gamma interferon (IFN-γ), a Th1 cytokine normally associated with classical Mφ activation and killing of intracellular pathogens. In this study, we provided several lines of evidence suggesting that IFN-γ-mediated parasite growth enhancement was associated withl-arginine transport via mouse cationic amino acid transporter 2B (mCAT-2B). (i) mRNA expression ofSlc7A2, the gene encoding for mCAT-2B, as well asl-arginine transport was increased in IFN-γ-treated Mφs. (ii) Supplementation ofl-arginine in Mφ cultures increased parasite growth. (iii) Parasite growth enhancement in wild-type Mφs was inhibited in the presence of nonmetabolizedl-arginine analogues. (iv) IFN-γ-mediated parasite growth was absent in Mφs derived from mCAT-2B-deficient mice. Although we detected a clear upregulation of mCAT-2B andl-arginine transport, no measurable iNOS or arginase activities were observed in IFN-γ-treated, infected Mφs. Together, these data suggest an involvement of a novell-arginine usage independent of iNOS and arginase activities during IFN-γ-mediated parasite growth enhancement. A possible role of mCAT-2B in supplyingl-arginine directly to the parasites for their proliferation is discussed.
Fatty Acids Isolated from
Toxoplasma gondii
Reduce Glycosylphosphatidylinositol-Induced Tumor Necrosis Factor Alpha Production through Inhibition of the NF-κB Signaling Pathway
Authors: Debierre-Grockiego Françoise , Rabi Khamran , Schmidt Jörg , Geyer Hildegard , Geyer Rudolf , Schwarz Ralph T.
Published: March 2007

Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and are known to induce inflammatory cytokines. However, we have previously shown that the family of six GPIs of
Toxoplasma gondii
extracted together from tachyzoites could not induce tumor necrosis factor alpha (TNF-α) secretion by macrophages, whereas GPIs individually separated from this extract by thin-layer chromatography (TLC) were able to stimulate the cells. In the present study we show that the TLC step makes it possible to eliminate inhibitors extracted together with the
T. gondii
GPIs. Among the non-GPI molecules we have isolated fatty acids able to inhibit the secretion of TNF-α induced by the
T. gondii
GPIs. Myristic and palmitic acids reduce the production of TNF-α through the inhibition of tyrosine phosphorylation of cytoplasmic proteins and the inhibition of NF-κB activation in a peroxisome proliferator-activated receptor-independent pathway and after a rapid entry into the cytoplasm of macrophages. GPIs are considered toxins inducing irreversible damage in the host, and fatty acids produced in parallel by the parasite could reduce the immune response, thus favoring the persistence of parasite infection.
Control of Quorum Sensing by a
Burkholderia pseudomallei
Multidrug Efflux Pump
Authors: Chan Ying Ying , Bian Hao Sheng , Tan Theresa May Chin , Mattmann Margrith E. , Geske Grant D. , Igarashi Jun , Hatano Tomomi , Suga Hiroaki , Blackwell Helen E. , Chua Kim Lee
Published: March 2007

The
Burkholderia pseudomallei
KHW quorum-sensing systems produced
N
-octanoyl-homoserine lactone,
N
-decanoyl-homoserine lactone,
N
-(3-hydroxy)-octanoyl-homoserine lactone,
N
-(3-hydroxy)-decanoyl-homoserine lactone,
N
-(3-oxo)-decanoyl-homoserine lactone, and
N
-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the
B. pseudomallei
BpeAB-OprB efflux pump.
Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling
Authors: Talaat Adel M. , Ward Sarah K. , Wu Chia-Wei , Rondon Elizabeth , Tavano Christine , Bannantine John P. , Lyons Rick , Johnston Stephen A.
Published: March 2007

Chronic tuberculosis represents a major health problem for one-third of the world's population today. A key question relevant to chronic tuberculosis is the physiological status of
Mycobacterium tuberculosis
during this important stage of infection. To examine the molecular bases of chronic tuberculosis and the role of host immunity in mycobacterial growth, we determined the mycobacterial transcriptional profiles during chronic and reactivation phases of murine tuberculosis using in vivo microarray analysis (IVMA). Following 28 days of aerosol infection, mycobacterial counts remained stable, although the bacilli were metabolically active with a 50% active transcriptome. The expression of genes involved in lipid and carbohydrate pathways was significantly enriched during the middle stage of chronic tuberculosis, suggesting a nutrient-rich microenvironment. A total of 137 genes were significantly regulated in mid-chronic tuberculosis (45 and 60 days) compared to an early stage (14 days) of infection. Additional sets of genes, including the virulence regulator
virS
, were up-regulated during the reactivation stage, indicating their possible roles in mycobacterial resurgence. Interestingly, a set of potential transcriptional regulators was significantly induced at the late stage of chronic tuberculosis. Bioinformatic analysis identified a large number of genes that could be regulated by one of the potential transcriptional regulators encoded by rv0348, including the
sigF
operon. Taken together, IVMA provided a better definition of the transcriptional machinery activated during chronic and reactivation stages of tuberculosis and identified a novel transcriptional regulator. A similar approach can be adopted to study key stages of intracellular pathogens.
Iron-Dependent RNA-Binding Activity of
Mycobacterium tuberculosis
Aconitase
Authors: Banerjee Sharmistha , Nandyala Ashok Kumar , Raviprasad Podili , Ahmed Niyaz , Hasnain Seyed E.
Published: March 2007

Cellular iron levels are closely monitored by iron regulatory and sensor proteins of
Mycobacterium tuberculosis
for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of
M. tuberculosis
aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that
M. tuberculosis
Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to
cis
-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays,
M. tuberculosis
Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3′ UTR of thioredoxin (
trxC
) and the 5′ UTR of the iron-dependent repressor and activator (
ideR
) of
M. tuberculosis. M. tuberculosis
Acn when reactivated with Fe
2+
functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of
M. tuberculosis
Acn are mutually exclusive. Our results demonstrate the bifunctional nature of
M. tuberculosis
Acn, pointing to its likely role in iron homeostasis.
Anaerobic Gene Expression in
Staphylococcus aureus
Authors: Fuchs Stephan , Pané-Farré Jan , Kohler Christian , Hecker Michael , Engelmann Susanne
Published: March 2007

An investigation of gene expression in
Staphylococcus aureus
after a switch from aerobic to anaerobic growth was initiated by using the proteomic and transcriptomic approaches. In the absence of external electron acceptors like oxygen or nitrate, an induction of glycolytic enzymes was observed. At the same time the amount of tricarboxylic acid cycle enzymes was very low. NAD is regenerated by mixed acid and butanediol fermentation, as indicated by an elevated synthesis level of fermentation enzymes like lactate dehydrogenases (Ldh1 and Ldh2), alcohol dehydrogenases (AdhE and Adh), α-acetolactate decarboxylase (BudA1), acetolactate synthase (BudB), and acetoin reductase (SACOL0111) as well as an accumulation of fermentation products as lactate and acetate. Moreover, the transcription of genes possibly involved in secretion of lactate (SACOL2363) and formate (SACOL0301) was found to be induced. The formation of acetyl-coenzyme A or acetyl-phosphate might be catalyzed by pyruvate formate lyase, whose synthesis was found to be strongly induced as well. Although nitrate was not present, the expression of genes related to nitrate respiration (NarH, NarI, and NarJ) and nitrate reduction (NirD) was found to be upregulated. Of particular interest, oxygen concentration might affect the virulence properties of
S. aureus
by regulating the expression of some virulence-associated genes such as
pls
,
hly
,
splC
and
splD
,
epiG
, and
isaB
. To date, the mechanism of anaerobic gene expression in
S. aureus
has not been fully characterized. In addition to
srrA
the mRNA levels of several other regulatory genes with yet unknown functions (e.g., SACOL0201, SACOL2360, and SACOL2658) were found to be upregulated during anaerobic growth, indicating a role in the regulation of anaerobic gene expression.
Biochemical Characterization and Physiological Properties ofEscherichia coliUDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase
Authors: Hervé Mireille , Boniface Audrey , Gobec Stanislav , Blanot Didier , Mengin-Lecreulx Dominique
Published: March 2007

The UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptidel-alanyl-γ-d-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme fromEscherichia coliwas purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptidel-Ala-d-Glu andl-Ala very poorly. Replacement ofmeso-diaminopimelic acid byl-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of theE. coli mplgene and/or theldcAgene encoding theld-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.
The Global Transcriptional Responses of
Bacillus anthracis
Sterne (34F
2
) and a Δ
sodA1
Mutant to Paraquat Reveal Metal Ion Homeostasis Imbalances during Endogenous Superoxide Stress
Authors: Passalacqua Karla D. , Bergman Nicholas H. , Lee Jung Yeop , Sherman David H. , Hanna Philip C.
Published: March 2007

Microarray analyses were conducted to evaluate the paraquat-induced global transcriptional response of
Bacillus anthracis
Sterne (34F
2
) to varying levels of endogenous superoxide stress. Data revealed that the transcription of genes putatively involved in metal/ion transport, bacillibactin siderophore biosynthesis, the glyoxalase pathway, and oxidoreductase activity was perturbed most significantly. A
B. anthracis
mutant lacking the superoxide dismutase gene
sodA1
(Δ
sodA1
) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the Δ
sodA1
mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and Δ
sodA1
cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The Δ
sodA1
mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in
B. anthracis
. A double mutant lacking both the
sodA1
and
sodA2
genes (Δ
sodA1
Δ
sodA2
) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between
sodA1
and
sodA2
, and a role for the
sod
genes in manganese homeostasis.
Transcription Antitermination by Translation Initiation Factor IF1
Authors: Phadtare Sangita , Kazakov Teymur , Bubunenko Mikhail , Court Donald L. , Pestova Tatyana , Severinov Konstantin
Published: March 2007

Bacterial translation initiation factor IF1 is an S1 domain protein that belongs to the oligomer binding (OB) fold proteins. Cold shock domain (CSD)-containing proteins such as CspA (the major cold shock protein of
Escherichia coli
) and its homologues also belong to the OB fold protein family. The striking structural similarity between IF1 and CspA homologues suggests a functional overlap between these proteins. Certain members of the CspA family of cold shock proteins act as nucleic acid chaperones: they melt secondary structures in nucleic acids and act as transcription antiterminators. This activity may help the cell to acclimatize to low temperatures, since cold-induced stabilization of secondary structures in nascent RNA can impede transcription elongation. Here we show that the
E. coli
translation initiation factor, IF1, also has RNA chaperone activity and acts as a transcription antiterminator in vivo and in vitro. We further show that the RNA chaperone activity of IF1, although critical for transcription antitermination, is not essential for its role in supporting cell growth, which presumably functions in translation. The results thus indicate that IF1 may participate in transcription regulation and that cross talk and/or functional overlap may exist between the Csp family proteins, known to be involved in transcription regulation at cold shock, and S1 domain proteins, known to function in translation.