bluuebunny/crossref_metadata_2025_split huggingface dataset:
cis
- and
trans
-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae
Authors: Subramanian Shoba , Woolford Carol A. , Desai Jigar V. , Lanni Frederick , Mitchell Aaron P. 
Published: August 2012

ABSTRACT

The Rim101/PacC pathway governs adaptation to alkaline pH in many fungi. Output of the pathway is mediated by transcription factors of the Rim101/PacC family, which are activated by proteolytic cleavage. The proteolytic complex includes scaffold protein Rim20 and endosome-associated subunits of the endosomal sorting complex required for transport (ESCRT). We provide here evidence that
Saccharomyces cerevisiae
Rim13, the protease that is implicated in Rim101 cleavage, is associated with the Rim20-ESCRT complex, and we investigate its regulation. Rim13-GFP is dispersed in cells grown in acidic medium but forms punctate foci when cells encounter alkaline conditions. A
vps4Δ
mutant, which accumulates elevated levels of endosomal ESCRT, also accumulates elevated levels of Rim13-GFP foci, independently of external pH. In the
vps4Δ
background, mutation of ESCRT subunit Snf7 or of Rim20 blocks the formation of Rim13 foci, and we found that Rim13 and Rim20 are colocalized. The Rim13 ortholog PalB of
Aspergillus nidulans
has been shown to undergo ESCRT and membrane association through an N-terminal MIT domain, but Rim13 orthologs in the
Saccharomyces
clade lack homology to this N-terminal region. Instead, there is a clade-limited C-terminal region, and we show that point mutations in this region prevent punctate localization and impair Rim13 function. We suggest that
RIM13
arose from its ancestral gene through two genome rearrangements. The ancestor lost the coding region for its MIT domain through a 5′ rearrangement and acquired the coding region for the
Saccharomyces
-specific functional equivalent through a 3′ rearrangement.
The AbrB2 Autorepressor, Expressed from an Atypical Promoter, Represses the Hydrogenase Operon To Regulate Hydrogen Production in Synechocystis Strain PCC6803
Authors: Dutheil Jérémy , Saenkham Panatda , Sakr Samer , Leplat Christophe , Ortega-Ramos Marcia , Bottin Hervé , Cournac Laurent , Cassier-Chauvat Corinne , Chauvat Franck 
Published: August 2012

ABSTRACT

We have thoroughly investigated the
abrB2
gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium
Synechocystis
strain PCC6803. We report that
abrB2
is expressed from an active but atypical promoter that possesses an extended −10 element (TGTAATAT) that compensates for the absence of a −35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended −10 promoter box and the absence of a −35 element are two well-conserved features in
abrB2
genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the
hox
operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the
hox
operon is weakly expressed even though it possesses the two sequences resembling canonical −10 and −35 promoter boxes. In both the AbrB2-repressed promoters of the
abrB2
gene and the
hox
operon, we found a repeated DNA motif [TT-(N
5
)-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the
abrB2
promoter. We think that our
abrB2
-deleted mutant with increased expression of the
hox
operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the
abrB2
gene and the
hox
operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in
Synechocystis
.
Origin and Fate of the 3′ Ends of Single-Stranded DNA Generated by Conjugal Transfer of Plasmid R1162
Authors: Becker Eric C. , Meyer Richard 
Published: August 2012

ABSTRACT

During conjugation, a single strand of DNA is cleaved at the origin of transfer (
oriT
) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5′-to-3′ direction, with the 3′ end likely extended by rolling-circle replication. The resulting, newly synthesized
oriT
must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at
oriT
, with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3′ ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved
oriT
within the strand. A model is presented where these internal
oriT
s bind and trap the relaxase molecule covalently bound to the 5′ end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.
Function and Regulation of Isoforms of Carbon Monoxide Dehydrogenase/Acetyl Coenzyme A Synthase in Methanosarcina acetivorans
Authors: Matschiavelli Nicole , Oelgeschläger Ellen , Cocchiararo Berardino , Finke Johannes , Rother Michael 
Published: August 2012

ABSTRACT

Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO
2
reduction and cleavage/synthesis of acetyl-CoA.
Methanosarcina acetivorans
C2A contains two very similar copies of a six-gene operon (
cdh
genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in
M. acetivorans
, mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of
cdh
disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are
bona fide
CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of
cdh1
, suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.
Minor Pilins of the Type IV Pilus System Participate in the Negative Regulation of Swarming Motility
Authors: Kuchma S. L. , Griffin E. F. , O'Toole G. A. 
Published: August 2012

ABSTRACTPseudomonas aeruginosaexhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either thepilWorpilXgene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations inpilWorpilXalso fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) orbifAmutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute toP. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems.
Phenotype Switching Is a Natural Consequence of Staphylococcus aureus Replication
Authors: Edwards Andrew M. 
Published: August 2012

ABSTRACT

The pathogen
Staphylococcus aureus
undergoes phenotype switching
in vivo
from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small-colony-variant (SCV) phenotype that is associated with persistence in host cells and tissues. However, it is not clear whether phenotype switching is the result of a constitutive process that is selected for under certain conditions or is triggered by particular environmental stimuli. Examination of cultures of diverse
S. aureus
strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV subpopulation that emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was reestablished by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure, however, phenotype switching was bidirectional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that
S. aureus
phenotype switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant subpopulation of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the
S. aureus
life cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population.
Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum
Authors: Jin Huanan , Nikolau Basil J. 
Published: August 2012

ABSTRACT

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the
Rhodospirillum rubrum
genome revealed by the occurrence of three homologous PHA polymerase genes (
phaC1
,
phaC2
, and
phaC3
).
In vitro
biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple
phaC
deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that
phaC2
contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast,
phaC3
is an insignificant contributor to PHA productivity, and
phaC1
, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C
5
and C
6
monomers. The
in vitro
characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of
R. rubrum
was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.
Reduced Na
+
Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB
Authors: Jakkula S. Vivek , Guan Lan 
Published: August 2012

ABSTRACT

The melibiose permease of
Salmonella enterica
serovar Typhimurium (MelB
St
) catalyzes symport of melibiose with Na
+
, Li
+
, or H
+
. Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na
+
-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (
V
max
) for melibiose transport in
Escherichia coli
DW2 and greatly decrease Na
+
affinity, indicating that intracellular release of Na
+
is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop
4-5
and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases
V
max
with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.
Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea
Authors: Tomita Hiroya , Yokooji Yuusuke , Ishibashi Takuya , Imanaka Tadayuki , Atomi Haruyuki 
Published: August 2012

ABSTRACT

Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4′-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon
Thermococcus kodakarensis
utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from
T. kodakarensis
. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable
k
cat
/

K
m

values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg
2+
, respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.
Development of a generic working definition of ‘supportive care’
Authors: Cramp Fiona , Bennett Michael I 
Published: August 2012

ContextThe term ‘supportive care’, despite everyday and widespread use, remains an ambiguous concept that lacks clarity. A generic definition would underpin the development of services and provide a basis for disease specific approaches to care.ObjectivesTo develop a generic working definition of supportive care that could be applied to a range of diseases and chronic illnesses.MethodsAn indepth review of the literature was carried out to identify existing generic and disease specific definitions and descriptions of supportive care. Following the review, a modified two-phase Delphi study was performed. Participants included experts in the area of supportive care identified during the review in addition to charities or their representatives. A draft definition was developed based upon the outcome of the Delphi study.ResultsThe literature review identified one brief generic definition of supportive care with no explanation of its origin. A further 17 disease specific definitions were identified, all relating to cancer. The review led to the development of 100 statements for inclusion in phase I of the modified Delphi study. 26 responses were received to phase I of the Delphi study and 17 responses to phase II. General agreement was received during the second stage and a final draft definition subsequently developed.ConclusionsThe resulting definition of supportive care might provide a basis for service development and could be further adapted to disease specific contexts. It would benefit from broader consultation to determine acceptability among a wider range of health professionals and service users.
Late effects and healthcare needs of survivors of allogeneic stem cell transplantation: a qualitative study
Authors: Hwang Jessica P , Roundtree Aimee K , Giralt Sergio A , Suarez-Almazor Maria 
Published: August 2012

PurposeThis qualitative study of survivors of allogeneic stem cell transplantation (SCT) for haematological malignancy explored attitudes about late effects of therapy, healthcare issues and information needs.MethodsWe conducted 12 indepth cognitive interviews and three focus groups of patients who had previously had SCT and were without recurrence of their primary disease. We used grounded theory methods, where themes emerged from consensus between cocoders. Health-related quality of life was assessed with the short-form 36 (SF-36).ResultsThe study included 22 patients (50% female; 95% white; mean age 47 years). The mean time from SCT was 5.2 years (±1.4 years). Most had low SF-36 scores. Participants discussed late effects of therapy, most commonly graft-versus-host disease, and how they impacted their quality of life. They reported frequent healthcare use and cancer screening after SCT and discussed problems affording care and interacting with insurance companies. Participants shared sources of health information (eg, preferring providers as their primary sources of information, but also learnt from websites, medical journals and peer experiences) and identified information barriers (eg, feeling ‘on their own’ insofar as they did not have targeted care for their needs), and expressed importance of anticipatory guidance regarding infertility. Overall, participants' personal issues and social influences impacted survivors' needs and attitudes.ConclusionsSCT survivors face continuing and lasting health effects. The factors impacting survivorship needs are complex and may be interrelated. Future research should study the affect of incorporating personal and social issues into existing clinical SCT programmes on survivors' quality of life.
Candidate Pathways for Promoting Differentiation or Quiescence of Oligodendrocyte Progenitor-like Cells in Glioma
Authors: Dougherty Joseph D. , Fomchenko Elena I. , Akuffo Afua A. , Schmidt Eric , Helmy Karim Y. , Bazzoli Elena , Brennan Cameron W. , Holland Eric C. , Milosevic Ana 
Published: August 2012

AbstractPlatelet-derived growth factor receptor alpha–positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. Human glioblastoma multiforme tumors often contain rapidly proliferating oligodendrocyte lineage transcription factor 2 (Olig2)-positive cells that resemble OPCs. In this study, we sought to identify candidate pathways that promote OPC differentiation or quiescence rather than proliferation. Gene expression profiling conducted in both normal murine OPCs and highly proliferative Olig2-positive glioma cells identified all the transcripts associated with the highly proliferative state of these cells and showed that among the various cell types found within the brain, Olig2-positive tumor cells are most similar to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have similar expression profiles in human tumors and were significantly enriched in genomic deletions, suggesting an antiproliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings show that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies. Cancer Res; 72(18); 4856–68. ©2012 AACR.
The Microculture-Kinetic (MiCK) Assay: The Role of a Drug-Induced Apoptosis Assay in Drug Development and Clinical Care
Authors: Bosserman Linda , Prendergast Franklyn , Herbst Roy , Fleisher Martin , Salom Emery , Strickland Steven , Raptis Anastasios , Hallquist Allan , Perree Mathieu , Rajurkar Swapnil , Karimi Misagh , Rogers Karl , Davidson Dirk , Willis Carl , Penalver Manuel , Homesley Howard , Burrell Matthew , Garrett Audrey , Rutledge James , Chernick Michael , Presant Cary A. 
Published: August 2012

Abstract
A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development. Cancer Res; 72(16); 3901–5. ©2012 AACR.