bluuebunny/crossref_metadata_2025_split huggingface dataset:
Contributions of ATP, GTP, and Redox State to Nutritional Stress Activation of theBacillus subtilisσBTranscription Factor
Authors: Zhang Shuyu , Haldenwang W. G. 
Published: November 2005

ABSTRACTThe general stress regulon ofBacillus subtilisis induced by activation of the σBtranscription factor. σBactivation occurs when one of two phosphatases responds to physical or nutritional stress to activate a positive σBregulator by dephosphorylation. The signal that triggers the nutritional stress phosphatase (RsbP) is unknown; however, RsbP activation occurs under culture conditions (glucose/phosphate starvation, azide or decoyinine treatment) that reduce the cell's levels of ATP and/or GTP. Variances in nucleotide levels in these instances may be coincidental rather than causal. RsbP carries a domain (PAS) that in some regulatory systems can respond directly to changes in electron transport, proton motive force, or redox potential, changes that typically precede shifts in high-energy nucleotide levels. The current work usesBacillus subtiliswith mutations in the oxidative phosphorylation and purine nucleotide biosynthetic pathways in conjunction with metabolic inhibitors to better define the inducing signal for RsbP activation. The data argue that a drop in ATP, rather than changes in GTP, proton motive force, or redox state, is the key to triggering σBactivation.
Adaptations of
Pseudomonas aeruginosa
to the Cystic Fibrosis Lung Environment Can Include Deregulation of
zwf
, Encoding Glucose-6-Phosphate Dehydrogenase
Authors: Silo-Suh Laura , Suh Sang-Jin , Phibbs Paul V. , Ohman Dennis E. 
Published: November 2005

ABSTRACT

Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid
Pseudomonas aeruginosa
strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in
zwf
, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a ∼90% reduction in alginate production in the mucoid, CF isolate,
P. aeruginosa
FRD1. The main regulator of alginate, sigma-22 encoded by
algT
(
algU
), plays a small but demonstrable role in the induction of
zwf
expression in
P. aeruginosa
. However, G6PDH activity and
zwf
expression were higher in FRD1 strains than in PAO1 strains. In PAO1,
zwf
expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1
zwf
expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of
zwf
was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident
P. aeruginosa
strain underwent an adaptive change that deregulated
zwf
expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to
P. aeruginosa
within the lung environment. Interestingly,
zwf
expression in
P. aeruginosa
was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by
P. aeruginosa
can include altered regulation of basic metabolic activities, including carbon catabolism.
Periplasmic Peptidyl Prolyl
cis-trans
Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in
Escherichia coli
Authors: Justice Sheryl S. , Hunstad David A. , Harper Jill Reiss , Duguay Amy R. , Pinkner Jerome S. , Bann James , Frieden Carl , Silhavy Thomas J. , Hultgren Scott J. 
Published: November 2005

ABSTRACT

In
Escherichia coli
, FkpA, PpiA, PpiD, and SurA are the four known periplasmic
cis
-
trans
prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the
cis
and
trans
states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the
surA
single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of
E. coli
and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.
Diverse Phenotypes Resulting from Polyphosphate Kinase Gene (
ppk1
) Inactivation in Different Strains of
Helicobacter pylori
Authors: Tan Shumin , Fraley Cresson D. , Zhang Maojun , Dailidiene Daiva , Kornberg Arthur , Berg Douglas E. 
Published: November 2005

ABSTRACT

Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for polyphosphate kinase 1 (
ppk1
) in strains of
Helicobacter pylori
, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies,
ppk1
inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Δ
ppk1
strain on F-12 agar and the inability to colonize mice of another were each due to
ppk1
inactivation. Thus, the importance of
ppk1
to
H. pylori
differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that
H. pylori
's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.
A Gyrase Mutant with Low Activity Disrupts Supercoiling at the Replication Terminus
Authors: Pang Zhenhua , Chen Ray , Manna Dipankar , Higgins N. Patrick 
Published: November 2005

ABSTRACTWhen a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. ThegyrB652mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42°C, strains with thegyrB652allele stop DNA replication, and at 37°C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42°C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a lowkcatcompared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.
Novel Virulence Gene of
Pseudomonas syringae
pv. tomato Strain DC3000
Authors: Preiter Karen , Brooks David M. , Penaloza-Vazquez Alejandro , Sreedharan Aswathy , Bender Carol L. , Kunkel Barbara N. 
Published: November 2005

ABSTRACT

Previously, we conducted a mutant screen of
Pseudomonas syringae
pv. tomato strain DC3000 to identify genes that contribute to virulence on
Arabidopsis thaliana
plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn
5
insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (
T
etR-like
v
i
r
ulence
r
egulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the
tvrR
promoter region,
tvrR
is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes.
tvrR
mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However,
tvrR
mutants do not cause disease symptoms on inoculated
A. thaliana
and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that
tvrR
mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on
A. thaliana
. Given that
tvrR
mutant strains are not defective for type III secretion or COR production,
tvrR
appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.
Polar Localization of a Soluble Methyl-Accepting Protein ofPseudomonas aeruginosa
Authors: Bardy Sonia L. , Maddock Janine R. 
Published: November 2005

ABSTRACTA soluble methyl-accepting chemotaxis protein (MCP) ofPseudomonas aeruginosa, McpS, showed polar localization by immunofluorescence microscopy. Overexpression of McpS resulted in a dominant-negative effect on chemotaxis and caused a loss of polar clustering of the general MCP population. The polar localization of a soluble MCP defines a third, and unexpected, paradigm for cellular MCP localization.
Streptococcus mutans
Murein Hydrolase
Authors: Catt Diana M. , Gregory Richard L. 
Published: November 2005

ABSTRACT

Allelic replacement of the C terminus of a
Streptococcus mutans
surface protein affects murein hydrolase activity. The targeted open reading frame encodes a 67-kDa protein (SmaA) with an N-terminal signal sequence and cleavage site, three 46-amino-acid (aa) direct repeats, and two 88-aa direct repeats. The identical autolytic profile was obtained using a sortase mutant (SrtA
−
).
Identification of an Aberrantly Spliced Form of HDMX in Human Tumors: A New Mechanism for HDM2 Stabilization
Authors: Giglio Simona , Mancini Francesca , Gentiletti Francesca , Sparaco Giorgia , Felicioni Lara , Barassi Fabio , Martella Carla , Prodosmo Andrea , Iacovelli Stefano , Buttitta Fiamma , Farsetti Antonella , Soddu Silvia , Marchetti Antonio , Sacchi Ada , Pontecorvi Alfredo , Moretti Fabiola 
Published: November 2005

Abstract
The HDMX protein is closely related to HDM2 with which it shares different structural domains, particularly the p53 binding domain and the ring finger domain, where the two HDM proteins interact. Several oncogenic forms derived from splicing of HDM2 have been described in cancer. This work aimed at investigating whether analogous forms of HDMX exist in human tumors. Here, we report the characterization of an aberrantly spliced form of HDMX, HDMX211, isolated from the thyroid tumor cell line, ARO. HDMX211 binds and stabilizes the HDM2 protein. Although it lacks the p53 binding domain, HDMX211 also stabilizes p53 by counteracting its degradation by HDM2. However, the resulting p53 is transcriptionally inactive and increasingly associated to its inhibitor HDM2. Expression of HDMX211 strongly enhances the colony-forming ability of human cells in the presence or absence of wild-type p53. Conversely, depletion of HDMX211 by small interfering RNA significantly reduces the growth of ARO cells and increases their sensitivity to chemotherapy. Screening of lung cancer biopsies shows the presence of HDMX211 in samples that overexpress HDM2 protein, supporting a pathologic role for this new protein. This is the first evidence of a variant form of HDMX that has oncogenic potential independently of p53. HDMX211 reveals a new mechanism for overexpression of the oncoprotein HDM2. Most interestingly, it outlines a possible molecular explanation for a yet unclarified tumor phenotype, characterized by simultaneous overexpression of HDM2 and wild-type p53.
Proteolysis of CCN1 by Plasmin: Functional Implications
Authors: Pendurthi Usha R. , Tran Tien T. , Post Marina , Rao L. Vijaya Mohan 
Published: November 2005

Abstract
Plasmin is shown to play a crucial role in many pathophysiologic processes primarily through its ability to degrade extracellular matrix (ECM) and/or mobilizing growth factors that are sequestered in the ECM. Cysteine-rich 61 (CCN1) is a matricellular protein of which expression is up-regulated in cancer and various vascular diseases. The present study was undertaken to investigate whether plasmin liberates CCN1 from the ECM and whether the released growth factor modulates endothelial cell migration. Treatment of breast carcinoma cells (MDA-MB-231) with plasmin released a truncated form of CCN1 (28 kDa) into the overlying medium. Experiments with recombinant CCN1 confirmed that plasmin effectively cleaves CCN1. Thrombin and other clotting/fibrinolytic proteases are ineffective in cleaving CCN1. Further studies revealed that the conditioned medium of plasmin-treated carcinoma cells supports endothelial cell migration and that antibodies specific to CCN1 blocked this enhancing effect. These data were the first to show that plasmin can liberate a pluripotent matrix signaling protein, CCN1, from the ECM. Because both CCN1 and the components of the plasmin generation system are present in tumor cells and a variety of other cells, the proteolysis of CCN1 by plasmin may play a role in many pathophysiologic processes, including tumor cell–mediated angiogenesis.
A Role for Polymerase η in the Cellular Tolerance to Cisplatin-Induced Damage
Authors: Albertella Mark R. , Green Catherine M. , Lehmann Alan R. , O'Connor Mark J. 
Published: November 2005

Abstract
Mutation of the POLH gene encoding DNA polymerase η (pol η) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol η to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol η can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol η. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol η expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol η expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol η is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol η foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol η in dealing with cisplatin-induced damage. Together, these data show that pol η represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.
Tocopherol-Associated Protein Suppresses Prostate Cancer Cell Growth by Inhibition of the Phosphoinositide 3-Kinase Pathway
Authors: Ni Jing , Wen Xingqiao , Yao Jorge , Chang Hong-Chiang , Yin Yi , Zhang Min , Xie Shaozhen , Chen Ming , Simons Brenna , Chang Philip , di Sant'Agnese Anthony , Messing Edward M. , Yeh Shuyuan 
Published: November 2005

Abstract
Epidemiologic studies suggested that vitamin E has a protective effect against prostate cancer. We showed here that tocopherol-associated protein (TAP), a vitamin E–binding protein, promoted vitamin E uptake and facilitated vitamin E antiproliferation effect in prostate cancer cells. Interestingly, without vitamin E treatment, overexpression of TAP in prostate cancer cells significantly suppressed cell growth; knockdown of endogenous TAP by TAP small interfering RNA (siRNA) in nonmalignant prostate HPr-1 cells increased cell growth. Further mechanism dissection studies suggested that the tumor suppressor function of TAP was via down-regulation of phosphoinositide 3-kinase (PI3K)/Akt signaling, but not by modulating cell cycle arrest or androgen receptor signaling. Immunoprecipitation results indicated that TAP inhibited the interaction of PI3K subunits, p110 with p85, and subsequently reduced Akt activity. Constitutively active Akt could negate the TAP-suppressive activity on prostate cancer cell growth. Moreover, stable transfection of TAP in LNCaP cells suppressed LNCaP tumor incidence and growth rate in nude mice. Furthermore, TAP mRNA and protein expression levels were significantly down-regulated in human prostate cancer tissue samples compared with benign prostate tissues as measured by reverse transcription-PCR, in situ hybridization, and immunohistochemistry. Together, our data suggest that TAP not only mediates vitamin E absorption to facilitate vitamin E antiproliferation effect in prostate cancer cells, but also functions like a tumor suppressor gene to control cancer cell viability through a non–vitamin E manner. Therefore, TAP may represent a new prognostic marker for prostate cancer progression.
HER2/neu-Induced Mammary Tumorigenesis and Angiogenesis Are Reduced in Cyclooxygenase-2 Knockout Mice
Authors: Howe Louise R. , Chang Sung-Hee , Tolle Kelly C. , Dillon Rachelle , Young Lawrence J.T. , Cardiff Robert D. , Newman Robert A. , Yang Peiying , Thaler Howard T. , Muller William J. , Hudis Clifford , Brown Anthony M.C. , Hla Timothy , Subbaramaiah Kotha , Dannenberg Andrew J. 
Published: November 2005

Abstract
The inducible prostaglandin synthase cyclooxygenase-2 (Cox-2) is overexpressed in ∼40% of human breast cancers and at higher frequencies in preinvasive ductal carcinoma in situ (DCIS). Cox-2 expression is particularly associated with overexpression of human epidermal growth factor receptor 2 (HER2/neu). To definitively interrogate the role of Cox-2 in mammary neoplasia, we have used a genetic approach, crossing Cox-2-deficient mice with a HER2/neu transgenic strain, MMTV/NDL. At 20 weeks of age, mammary glands from virgin MMTV/NDL females contained multiple focal tumors, or mammary intraepithelial neoplasias, which histologically resembled human DCIS. Mammary tumor multiplicity and prostaglandin E2 (PGE2) levels were significantly decreased in Cox-2 heterozygous and knockout animals relative to Cox-2 wild-type controls. Notably, the proportion of larger tumors was decreased in Cox-2-deficient mice. HER2/neu-induced mammary hyperplasia was also substantially reduced in Cox-2 null mice. Additionally, mammary glands from Cox-2 knockout mice exhibited a striking reduction in vascularization, and expression of proangiogenic genes was correspondingly reduced. Decreased vascularization was observed both in dysplastic and normal-appearing regions of Cox-2-null mammary glands. Our data provide the first genetic evidence that Cox-2 contributes to HER2/neu-induced mammary tumorigenesis. This finding may help to explain the reduced risk of breast cancer associated with regular use of nonsteroidal anti-inflammatory drugs.
Mutations of C-RAF Are Rare in Human Cancer because C-RAF Has a Low Basal Kinase Activity Compared with B-RAF
Authors: Emuss Victoria , Garnett Mathew , Mason Clive ,   The Cancer Genome Project, Marais Richard 
Published: November 2005

Abstract
The protein kinase B-RAF is mutated in ∼8% of human cancers. Here we show that presumptive mutants of the closely related kinase, C-RAF, were detected in only 4 of 545 (0.7%) cancer cell lines. The activity of two of the mutated proteins is not significantly different from that of wild-type C-RAF and these variants may represent rare human polymorphisms. The basal and B-RAF–stimulated kinase activities of a third variant are unaltered but its activation by RAS is significantly reduced, suggesting that it may act in a dominant-negative manner to modulate pathway signaling. The fourth variant has elevated basal kinase activity and is hypersensitive to activation by RAS but does not transform mammalian cells. Furthermore, when we introduce the equivalent of the most common cancer mutation in B-RAF (V600E) into C-RAF, it only has a weak effect on kinase activity and does not convert C-RAF into an oncogene. This lack of activation occurs because C-RAF lacks a constitutive charge within a motif in the kinase domain called the N-region. This fundamental difference in RAF isoform regulation explains why B-RAF is frequently mutated in cancer whereas C-RAF mutations are rare.
Asbestos-Induced Lung Inflammation and Epithelial Cell Proliferation Are Altered in Myeloperoxidase-Null Mice
Authors: Haegens Astrid , van der Vliet Albert , Butnor Kelly J. , Heintz Nicholas , Taatjes Douglas , Hemenway David , Vacek Pamela , Freeman Bruce A. , Hazen Stanley L. , Brennan Marie Luise , Mossman Brooke T. 
Published: November 2005

Abstract
Asbestos fibers are carcinogens causing oxidative stress and inflammation, but the sources and ramifications of oxidant production by asbestos are poorly understood. Here, we show that inhaled chrysotile asbestos fibers cause increased myeloperoxidase activity in bronchoalveolar lavage fluids (BALF) and myeloperoxidase immunoreactivity in epithelial cells lining distal bronchioles and alveolar ducts, sites of initial lung deposition of asbestos fibers. In comparison with sham mice, asbestos-exposed myeloperoxidase-null (MPO−/−) and normal (MPO+/+) mice exhibited comparable increases in polymorphonuclear leukocytes, predominately neutrophils, in BALF after 9 days of asbestos inhalation. Differential cell counts on BALF revealed decreased proportions of macrophages and increased lymphocytes in all mice exposed to asbestos, but numbers were decreased overall in asbestos-exposed myeloperoxidase-null versus normal mice. Asbestos-associated lung inflammation in myeloperoxidase-null mice was reduced (P ≤ 0.05) in comparison with normal asbestos-exposed mice at 9 days. Decreased lung inflammation in asbestos-exposed myeloperoxidase-null mice at 9 days was accompanied by increases (P ≤ 0.05) in Ki-67- and cyclin D1-positive immunoreactive cells, markers of cell cycle reentry, in the distal bronchiolar epithelium. Asbestos-induced epithelial cell proliferation in myeloperoxidase-null mice at 30 days was comparable to that found at 9 days. In contrast, inflammation and epithelial cell proliferation in asbestos-exposed normal mice increased over time. These results support the hypothesis that myeloperoxidase status modulates early asbestos-induced oxidative stress, epithelial cell proliferation, and inflammation.