bluuebunny/crossref_metadata_2025_split huggingface dataset:
The N-Terminal Domain of OmpATb Is Required for Membrane Translocation and Pore-Forming Activity in Mycobacteria
Authors: Alahari Anuradha , Saint Nathalie , Campagna Sylvie , Molle Virginie , Molle Gérard , Kremer Laurent 
Published: June 2007

ABSTRACT

OmpATb is the prototype of a new family of porins in
Mycobacterium tuberculosis
and
Mycobacterium bovis
BCG. Although the pore-forming activity of this protein has been clearly established by using recombinant protein produced in
Escherichia coli
, characterization of the native porin has been hampered by the scarce amount of protein present in the
M. tuberculosis
detergent extracts. To this aim, we have developed a protocol to overproduce and obtain high yields of OmpATb in both
Mycobacterium smegmatis
and
M. bovis
BCG. The protein could be extracted and purified from the cell wall fraction and subsequently used for analysis of the pore-forming activity in multichannel and single-channel conductance experiments. Our results indicate that OmpATb produced in mycobacteria presents an average conductance value of 1,600 ± 100 pS, slightly higher than that of OmpATb produced in
E. coli
, suggesting the occurrence of OmpATb in a highly ordered organization within the mycobacterial cell wall. In contrast to OmpATb, a truncated form lacking the first 72 amino acids (OmpATb
73-326
) was essentially found in the cytosol and was not active in planar lipid bilayers. This suggested that the N-terminal domain of OmpATb could participate in targeting of OmpATb to the cell wall. This was further confirmed by analyzing
M. smegmatis
clones expressing a chimeric protein consisting of a fusion between the N-terminal domain of OmpATb and the
E. coli
PhoA reporter. The present study shows for the first time that the N terminus of OmpATb is required for targeting the porin to the cell wall and also appears to be essential for its pore-forming activity.
Determinants of Entry Exclusion within Eex and TraG Are Cytoplasmic
Authors: Marrero Joeli , Waldor Matthew K. 
Published: June 2007

ABSTRACT
We report here functional and topological analyses of TraG and Eex, the donor and recipient cell inner membrane proteins that mediate entry exclusion in the SXT/R391 family of integrative conjugative elements. We found that the exclusion-determining regions of the Eex variants EexS (SXT) and EexR (R391) are located in distinct yet overlapping regions of the proteins. Unexpectedly, the carboxyl-terminal regions of TraG and Eex, which contain the residues essential for exclusion activity and specificity, were found to localize in the cell cytoplasm. These observations suggest that complex topological rearrangements of conjugative proteins must occur during mating to enable these domains to interact.
Comparative Analysis of the Regulation ofrovAfrom the Pathogenic Yersiniae
Authors: Lawrenz Matthew B. , Miller Virginia L. 
Published: June 2007

ABSTRACTRovA is a MarR/SlyA-type regulator that mediates the transcription ofinvinYersinia enterocoliticaandY. pseudotuberculosis. InY. pseudotuberculosis,rovAtranscription is controlled primarily by H-NS and RovA, which bind to similar regions within therovApromoter. At 37°C,rovAtranscription is repressed by H-NS. Transcription ofrovAresults when RovA relieves H-NS-mediated repression. The region of therovApromoter that H-NS and RovA bind is not conserved in theY. enterocoliticapromoter. Using green fluorescent protein reporters, we determined that theY. enterocolitica rovA(rovAYent) promoter is weaker than theY. pseudotuberculosispromoter. However, despite the missing H-NS/RovA binding site in therovAYentpromoter, H-NS and RovA are still involved in the regulation ofrovAYent. DNA binding studies suggest that H-NS and RovA bind with a higher affinity to theY. pseudotuberculosis/Y. pestis rovA(rovAYpstb/Ypestis) promoter than to therovAYentpromoter. Furthermore, H-NS appears to bind to two regions in a cooperative fashion within therovAYentpromoter that is not observed with therovAYpstb/Ypestispromoter. Finally, using a transposon mutagenesis approach, we identified a new positive regulator ofrovAinY. enterocolitica, LeuO. InEscherichia coli, LeuO regulates gene expression via changes in levels of RpoS and H-NS, but LeuO-mediated regulation ofrovAYentappears to be independent of either of these two proteins. Together, these data demonstrate that while therovAregulatory factors are conserved inYersinia, divergence ofY. enterocoliticaandY. pseudotuberculosis/Y. pestisduring evolution has resulted in modifications in the mechanisms that are responsible for controllingrovAtranscription.
Phosphorylcholine Expression by NontypeableHaemophilus influenzaeCorrelates with Maturation of Biofilm Communities In Vitro and In Vivo
Authors: Hong Wenzhou , Pang Bing , West-Barnette Shayla , Swords W. Edward 
Published: June 2007

ABSTRACTNontypeableHaemophilus influenzae(NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019licD) or were constitutively locked in the PCho-positive phase (NTHI 2019licON). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019licON. The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.
Oxidative Stress Evokes a Metabolic Adaptation That Favors Increased NADPH Synthesis and Decreased NADH Production in
Pseudomonas fluorescens
Authors: Singh Ranji , Mailloux Ryan J. , Puiseux-Dao Simone , Appanna Vasu D. 
Published: June 2007

ABSTRACT

The fate of all aerobic organisms is dependent on the varying intracellular concentrations of NADH and NADPH. The former is the primary ingredient that fuels ATP production via oxidative phosphorylation, while the latter helps maintain the reductive environment necessary for this process and other cellular activities. In this study we demonstrate a metabolic network promoting NADPH production and limiting NADH synthesis as a consequence of an oxidative insult. The activity and expression of glucose-6-phosphate dehydrogenase, malic enzyme, and NADP
+
-isocitrate dehydrogenase, the main generators of NADPH, were markedly increased during oxidative challenge. On the other hand, numerous tricarboxylic acid cycle enzymes that supply the bulk of intracellular NADH were significantly downregulated. These metabolic pathways were further modulated by NAD
+
kinase (NADK) and NADP
+
phosphatase (NADPase), enzymes known to regulate the levels of NAD
+
and NADP
+
. While in menadione-challenged cells, the former enzyme was upregulated, the phosphatase activity was markedly increased in control cells. Thus, NADK and NADPase play a pivotal role in controlling the cross talk between metabolic networks that produce NADH and NADPH and are integral components of the mechanism involved in fending off oxidative stress.
Identification of the RsmG Methyltransferase Target as 16S rRNA Nucleotide G527 and Characterization of
Bacillus subtilis rsmG
Mutants
Authors: Nishimura Kenji , Johansen Shanna K. , Inaoka Takashi , Hosaka Takeshi , Tokuyama Shinji , Tahara Yasutaka , Okamoto Susumu , Kawamura Fujio , Douthwaite Stephen , Ochi Kozo 
Published: June 2007

ABSTRACT

The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of
Bacillus subtilis
(corresponding to G527 in
Escherichia coli
). Disruption of
rsmG
resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the
rsmG
mutant and parent strains under the various culture conditions examined.
B. subtilis rsmG
mutants emerged spontaneously at a relatively high frequency, 10
−6
. Importantly, in the
rsmG
mutant background, high-level-streptomycin-resistant
rpsL
(encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in
rpsL
more varied than that obtained by selection of the wild-type strain.
Physiological and Expression Analyses of
Agrobacterium tumefaciens trxA
, Encoding Thioredoxin
Authors: Vattanaviboon Paiboon , Tanboon Weerachai , Mongkolsuk Skorn 
Published: June 2007

ABSTRACT

Exposure of
Agrobacterium tumefaciens
to menadione, cumene hydroperoxide, and diamide strongly induced
trxA
expression. The
trxA
mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome
c
oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H
2
O
2
resistance phenotype.
pcd
Mutants of
Streptomyces clavuligerus
Still Produce Cephamycin C
Authors: Alexander Dylan C. , Anders Cecilia L. , Lee Linda , Jensen Susan E. 
Published: June 2007

ABSTRACT

Biosynthesis of cephamycin C in
Streptomyces clavuligerus
involves the initial conversion of lysine to α-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the
lat
gene causes complete loss of cephamycin production,
pcd
mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by
pcd
mutants could be restored to wild-type levels either by supplementation of the growth medium with α-aminoadipic acid or by complementation of the mutation with an intact copy of the
pcd
gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second
pcd
gene. Furthermore, cell extracts from
pcd
mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that
S. clavuligerus
must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis.
Identification of Differentially Regulated Splice Variants and Novel Exons in Glial Brain Tumors Using Exon Expression Arrays
Authors: French Pim J. , Peeters Justine , Horsman Sebastiaan , Duijm Elza , Siccama Ivar , van den Bent Martin J. , Luider Theo M. , Kros Johan M. , van der Spek Peter , Sillevis Smitt Peter A. 
Published: June 2007

Abstract
Aberrant splice variants are involved in the initiation and/or progression of glial brain tumors. We therefore set out to identify splice variants that are differentially expressed between histologic subgroups of gliomas. Splice variants were identified using a novel platform that profiles the expression of virtually all known and predicted exons present in the human genome. Exon-level expression profiling was done on 26 glioblastomas, 22 oligodendrogliomas, and 6 control brain samples. Our results show that Human Exon arrays can identify subgroups of gliomas based on their histologic appearance and genetic aberrations. We next used our expression data to identify differentially expressed splice variants. In two independent approaches, we identified 49 and up to 459 exons that are differentially spliced between glioblastomas and oligodendrogliomas, a subset of which (47% and 33%) were confirmed by reverse transcription-PCR (RT-PCR). In addition, exon level expression profiling also identified &gt;700 novel exons. Expression of ∼67% of these candidate novel exons was confirmed by RT-PCR. Our results indicate that exon level expression profiling can be used to molecularly classify brain tumor subgroups, can identify differentially regulated splice variants, and can identify novel exons. The splice variants identified by exon level expression profiling may help to detect the genetic changes that cause or maintain gliomas and may serve as novel treatment targets. [Cancer Res 2007;67(12):5635–8]
Acquired Resistance to Erlotinib in A-431 Epidermoid Cancer Cells Requires Down-regulation of MMAC1/PTEN and Up-regulation of Phosphorylated Akt
Authors: Yamasaki Fumiyuki , Johansen Mary J. , Zhang Dongwei , Krishnamurthy Savitri , Felix Edward , Bartholomeusz Chandra , Aguilar Richard J. , Kurisu Kaoru , Mills Gordon B. , Hortobagyi Gabriel N. , Ueno Naoto T. 
Published: June 2007

Abstract
Erlotinib (Tarceva), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has clinical activity in advanced lung cancer, but disease that initially responds to erlotinib eventually progresses. The mechanism of this acquired resistance is unclear. We established two erlotinib-resistant pools of A-431 cells, a well-characterized epidermoid cancer cell line that constitutively overexpresses EGFR and is sensitive to erlotinib, by continuous exposure to erlotinib over a 6-month period. The extent of EGFR gene amplification or mutation of the EGFR tyrosine kinase domain was not altered in the resistant cells. Intracellular erlotinib concentrations, determined by liquid chromatography-tandem mass spectrometry, were almost the same in all three cell lines. Immunoprecipitation with EGFR antibody followed by detection with phosphotyrosine antibody revealed that erlotinib effectively reduced EGFR phosphorylation in both parental cells and resistant cells. Erlotinib induced mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) and suppressed phosphorylated Akt (Ser473) but not in the erlotinib-resistant cells. Overexpression of MMAC1/PTEN by transfection with Ad.MMAC1/PTEN or by pharmacologic suppression of Akt activity restored erlotinib sensitivity in both resistant pools. Further, transfection of parental A-431 cells with constitutively active Akt was sufficient to cause resistance to erlotinib. We propose that acquired erlotinib resistance associated with MMAC1/PTEN down-regulation and Akt activation could be overcome by inhibitors of signaling through the phosphatidylinositol 3-kinase pathway. [Cancer Res 2007;67(12):5779–88]
Effects of Sustained Antiangiogenic Therapy in Multistage Prostate Cancer in TRAMP Model
Authors: Isayeva Tatyana , Chanda Diptiman , Kallman Lisa , Eltoum Isam-Eldin A. , Ponnazhagan Selvarangan 
Published: June 2007

AbstractAntiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus–(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease. [Cancer Res 2007;67(12):5789–97]
Dactylone Inhibits Epidermal Growth Factor–Induced Transformation and Phenotype Expression of Human Cancer Cells and Induces G1-S Arrest and Apoptosis
Authors: Fedorov Sergey N. , Shubina Larisa K. , Bode Ann M. , Stonik Valentin A. , Dong Zigang 
Published: June 2007

Abstract
The marine natural chamigrane-type sesquiterpenoid, dactylone, is closely related to secondary metabolites of some edible species of red algae. In the present study, the effect of dactylone was tested on the mouse skin epidermal JB6 P+ Cl41 cell line and its stable transfectants as well as on several human tumor cell lines, including lung (H460), colon (HCT-116), and skin melanomas (SK-MEL-5 and SK-MEL-28). This natural product was effective at nontoxic doses as a cancer-preventive agent, which exerted its actions, at least in part, through the inhibition of cyclin D3 and Cdk4 expression and retinoblastoma tumor suppressor protein (Rb) phosphorylation. The inhibition of these cell cycle components was followed by cell cycle arrest at the G1-S transition with subsequent p53-independent apoptosis. Therefore, these data showed that application of dactylone and related compounds may lead to decreased malignant cell transformation and/or decreased tumor cell proliferation. [Cancer Res 2007;67(12):5914–20]
Loss of Xeroderma Pigmentosum C (Xpc) Enhances Melanoma Photocarcinogenesis in Ink4a-Arf–Deficient Mice
Authors: Yang Guang , Curley David , Bosenberg Marcus W. , Tsao Hensin 
Published: June 2007

Abstract
Despite an extensive body of evidence linking UV radiation and melanoma tumorigenesis, a clear mechanistic understanding of this process is still lacking. Because heritable mutations in both INK4a and the nucleotide excision repair (NER) pathway predispose individuals to melanoma development, we set out to test the hypothesis that abrogation of NER, by deletion of the xeroderma pigmentosum C (Xpc) gene, will heighten melanoma photocarcinogenesis in an Ink4a-Arf–deficient background. Experimentally, we generated a strain of mice doubly deficient in Xpc and Ink4a-Arf and subjected wild-type, Xpc−/−Ink4a-Arf+/+, Xpc−/−Ink4a-Arf−/−, and Xpc+/+Ink4a-Arf−/− mice to a single neonatal (day P3) dose of UVB without additional chemical promotion. Indeed, there was a significant increase in the development of dermal spindle/epithelioid cell melanomas in Xpc−/−Ink4a-Arf−/− mice when compared with Xpc+/+Ink4a-Arf−/− mice (P = 0.005); wild-type and Xpc−/−Ink4a-Arf+/+ mice failed to develop tumors. These neoplasms bore a striking histologic resemblance to melanomas that arise in the Tyr-vHRAS/Ink4a-Arf−/− context and often expressed melanocyte differentiation marker Tyrp1, thus supporting their melanocytic origination. All strains, except wild-type mice, developed pigmented and non-pigmented epidermal-derived keratinocytic cysts, whereas Xpc+/+Ink4a-Arf−/− mice exhibited the greatest propensity for squamous cell carcinoma development. We then screened for NRas, HRas, Kras, and BRaf mutations in tumor tissue and detected a higher frequency of rare KrasQ61 alterations in tumors from Xpc−/−Ink4a-Arf−/− mice compared with Xpc+/+Ink4a-Arf−/− mice (50% versus 7%, P = 0.033). Taken together, results from this novel UV-inducible melanoma model suggest that NER loss, in conjunction with Ink4a-Arf inactivation, can drive melanoma photocarcinogenesis possibly through signature Kras mutagenesis. [Cancer Res 2007;67(12):5649–57]
Antagonism of HOX/PBX Dimer Formation Blocks the In vivo Proliferation of Melanoma
Authors: Morgan Richard , Pirard Patricia Macanas , Shears Liesl , Sohal Jastinder , Pettengell Ruth , Pandha Hardev S. 
Published: June 2007

Abstract
Malignant melanoma is a cancer that arises from melanocyte cells in a complex but well-studied process, and which can only be successfully treated prior to metastasis as it is highly resistant to conventional therapies. A number of recent reports have indicated that members of the HOX family of homeodomain-containing transcription factors are deregulated in melanoma, and may actually be required to maintain proliferation. In this report, we describe the use of a novel, cell-permeable antagonist of the interaction between HOX proteins and PBX, a second homeodomain-containing transcription factor that modifies HOX activity. This antagonist can block the growth of murine B16 cells and trigger apoptosis both in vitro and in vivo when administered to mice with flank tumors. [Cancer Res 2007;67(12):5806–13]
Targeting Mutant (V600E)B-Rafin Melanoma Interrupts Immunoediting of Leukocyte Functions and Melanoma Extravasation
Authors: Liang Shile , Sharma Arati , Peng Hsin-Hsin , Robertson Gavin , Dong Cheng 
Published: June 2007

AbstractPolymorphonuclear neutrophils (PMN) facilitate melanoma cell extravasation under dynamic flow conditions by the binding of intercellular adhesion molecule-1 (ICAM-1) on melanoma cells to β2 integrins on PMNs, which is mediated by endogenously produced chemokine interleukin 8 (IL-8) from the tumor microenvironment. However, little is known about the role of B-Raf, the most mutated gene in malignant melanomas, in this process. In this study, we investigated the functional importance of B-Raf in melanoma extravasation by using short interfering RNA to reduce expression/activity of mutant V600EB-Raf in melanoma. Results indicated that knockdown of mutant V600EB-Raf inhibited melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inhibition of V600EB-Raf significantly reduced the constitutive secretion of IL-8 from melanoma cells as well as the capacity of endogenous IL-8 production from the melanoma-PMN microenvironment. Furthermore, a reduction in ICAM-1 expression on melanoma cells was detected following mutant V600EB-Raf knockdown. Together, these results suggest that targeting mutant V600EB-Raf reduces melanoma cell extravasation by decreasing IL-8 production and interrupting ICAM-1-β2 integrin binding of melanoma cells to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant V600EB-Raf to inhibit melanoma extravasation and subsequent metastasis development. [Cancer Res 2007;67(12):5814–20]