bluuebunny/crossref_metadata_2025 huggingface dataset:
Alteration of the Protein Kinase Binding Domain Enhances Function of the
Saccharomyces cerevisiae
Molecular Chaperone Cdc37
Authors: Ren Min , Santhanam Arti , Lee Paul , Caplan Avrom , Garrett Stephen
Published: June 2007

Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative “protein kinase binding domain” of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele,
tpk2
(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the
tpk2
(Ts) growth defect is also alleviated by Cdc37 overproduction, the
CDC37
dominant mutants contain wild-type Cdc37 protein levels. In addition,
Saccharomyces cerevisiae
Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.
Flagellar Elongation and Gene Expression inChlamydomonas reinhardtii
Authors: Periz Goran , Dharia Darshita , Miller Steven H. , Keller Laura R.
Published: June 2007

Lithium (Li+) affects the physiology of cells from a broad range of organisms including plants and both vertebrate and invertebrate animals. Although its effects result presumably from changes in gene expression elicited by its interaction with intracellular signal transduction pathways, the molecular mechanisms of Li+action are not well understood. The biflagellate green algaChlamydomonas reinhardtiiis an ideal genetic model for the integration of the effects on Li+on signal transduction, gene expression, and aspects of flagellar biogenesis. Li+causesC. reinhardtiiflagella to elongate to ∼1.4 times their normal length and blocks flagellar motility (S. Nakamura, H. Tabino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). We report here that Li+treatment increases the abundance of several flagellar mRNAs, including α- and β-tubulin and pcf3-21. Li+-induced flagellar gene expression occurs in cells pretreated with cycloheximide, suggesting that the abundance change is a response that does not require new protein synthesis. Deletion analysis of the flagellar α1-tubulin gene promoter showed that sequences necessary for Li+-induced expression differed from those for acid shock induction and contain a consensus binding site for CREB/ATF and AP-1 transcription factors. These studies suggest potential promoter elements, candidate factors, and signal transduction pathways that may coordinate theC. reinhardtiicellular response to Li+.
Integration of Rotation and Piston Motions in Coiled-Coil Signal Transduction
Authors: Gao Rong , Lynn David G.
Published: June 2007

A coordinated response to a complex and dynamic environment requires an organism to simultaneously monitor and interpret multiple signaling cues. In bacteria and some eukaryotes, environmental responses depend on the histidine autokinases (HKs). For example, VirA, a large integral membrane HK fromAgrobacterium tumefaciens, regulates the expression of virulence genes in response to signals from multiple molecular classes (phenol, pH, and sugar). The ability of this pathogen to perceive inputs from different known host signals within a single protein receptor provides an opportunity to understand the mechanisms of signal integration. Here we exploited the conserved domain organization of the HKs and engineered chimeric kinases to explore the signaling mechanisms of phenol sensing and pH/sugar integration. Our data implicate a piston-assisted rotation of coiled coils for integration of multiple inputs and regulation of critical responses during pathogenesis.
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

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

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

RovA 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

NontypeableHaemophilus 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

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

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

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

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.
Early changes in coronary artery wall structure detected by microcomputed tomography in experimental hypercholesterolemia
Authors: Zhu Xiang-Yang , Bentley Michael D. , Chade Alejandro R. , Ritman Erik L. , Lerman Amir , Lerman Lilach O.
Published: June 2007

Changes in the structure of the artery wall commence shortly after exposure to cardiovascular risk factors, such as hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful in evaluation of the factors that instigate atherosclerosis and its pathomechanisms. The present study tested the hypothesis that early morphological changes in coronary arteries of hypercholesterolemic (HC) pigs can be detected using the novel X-ray contrast agent OsO4 and three-dimensional micro-computed tomography (CT). Two groups of pigs were studied after they were fed a normal or an HC (2% cholesterol) diet for 12 wk. Hearts were harvested, coronary arteries were injected with 1% OsO4 solution, and cardiac samples (6-μm-thick) were scanned by micro-CT. Layers of the epicardial coronary artery wall, early lesions, and perivascular OsO4 accumulation were determined. Leakage of OsO4 from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor in corresponding histological cross sections. OsO4 enhanced the visualization of coronary artery wall layers and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO4 in HC was correlated with increased vascular endothelial growth factor expression and suggested increased microvascular permeability. The use of OsO4 as a contrast agent in micro-CT allows three-dimensional visualization of coronary artery wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.
Differential sympathetic outflow elicited by active muscle in rats
Authors: Koba Satoshi , Xing Jihong , Sinoway Lawrence I. , Li Jianhua
Published: June 2007

The present study was undertaken to test the hypothesis that activation of the muscle reflex elicits less sympathetic activation in skeletal muscle than in internal organs. In decerebrate rats, we examined renal and lumbar (mainly innervating hindlimb blood vessels) sympathetic nerve activities (RSNA and LSNA, respectively) during 1 min of 1) repetitive (1- to 4-s stimulation-to-relaxation) contraction of the triceps surae muscle, 2) repetitive tendon stretch, and 3) repetitive contraction with hindlimb circulatory occlusion. During these interventions, RSNA and LSNA responded synchronously as tension developed. The increase was greater in RSNA than in LSNA [+51 ± 14 vs. +24 ± 5% ( P < 0.05) with contraction, +46 ± 8 vs. +17 ± 4% ( P < 0.05) with stretch, +76 ± 20 vs. 39 ± 7% ( P < 0.05) with contraction during occlusion] during all three interventions: repetitive contraction ( n = 10, +508 ± 48 g tension from baseline), tendon stretch ( n = 12, +454 ± 34 g), and contraction during occlusion ( n = 9, +473 ± 33 g). Additionally, hindlimb circulatory occlusion significantly enhanced RSNA and LSNA responses to contraction. These data demonstrate that RSNA responses to muscle contraction and stretch are greater than LSNA responses. We suggest that activation of the muscle afferents induces the differential sympathetic outflow that is directed toward the kidney as opposed to the limbs. This differential outflow contributes to the distribution of cardiac output observed during exercise. We further suggest that as exercise proceeds, muscle metabolites produced in contracting muscle sensitize muscle afferents and enhance sympathetic drive to limbs and renal beds.
Predisposition to tetraploidy in pulmonary vascular smooth muscle cells derived from the Eker rats
Authors: Gui Yu , He Gordon H. , Walsh Michael P. , Zheng Xi-Long
Published: June 2007

Somatic mutations in the tuberous sclerosis complex-2 (TSC2) gene are associated with pulmonary lymphangioleiomyomatosis (LAM), a disorder characterized by benign lesions of smooth muscle and/or smooth muscle-like cells in the lung. However, the cellular mechanisms underlying LAM disease are largely unknown. Given that the TSC2 gene product tuberin is involved in the regulation of cell growth and proliferation, the present study was designed to investigate the potential roles of TSC2 in regulation of the cell cycle. We studied cell cycle profiles of pulmonary vascular smooth muscle cells (SMCs) derived from Eker rats (Tsc2+/EK), a genetic model carrying a germline insertional deletion in one copy of the Tsc2 gene, and the wild-type rats (Tsc2+/+), a noncarrier counterpart. We found that Tsc2+/EK, but not Tsc2+/+, SMCs displayed increases in cells with ≥4N DNA content (≥4N cells) and in the bromodeoxyuridine (BrdU) incorporation of ≥4N cells. Centrosome number was also increased in Tsc2+/EK SMCs, but the mitotic index was comparable between Tsc2+/+ and Tsc2+/EK SMCs. Furthermore, Tsc2+/EK SMCs showed elevated phosphorylation of p70S6K and increased expression of cell cycle regulatory proteins Cdk1, cyclin B, Cdk2, and cyclin E. Inhibition of the mammalian target of rapamycin (mTOR) pathway by rapamycin not only inhibited the phosphorylation of p70S6K and the expression of cell cycle regulatory proteins but also reduced accumulation of ≥4N cells and BrdU incorporation of >4N cells. Therefore, our results demonstrate that Tsc2+/EK SMCs are predisposed to undergo tetraploidization, involving activation of the mTOR pathway. These findings suggest an important role of Tsc2 in regulation of the cell cycle.
Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage
Authors: Pagano Alessandra , Métrailler-Ruchonnet Isabelle , Aurrand-Lions Michel , Lucattelli Monica , Donati Yves , Argiroffo Constance Barazzone
Published: June 2007

Oxygen-based therapies expose lung to elevated levels of ROS and induce lung cell damage and inflammation. Injured cells are replaced through increased proliferation and differentiation of epithelial cells and fibroblasts. Failure to modulate these processes leads to excessive cell proliferation, collagen deposition, fibrosis, and chronic lung disease. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. In this study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure. We exposed PARP-1 −/− and wild-type mice for 64 h to 100% hyperoxia and let them recover in air for 5–21 days. PARP-1-deficient mice exhibited significantly higher lung cell hyperplasia and proliferation than PARP-1 +/+ animals after 5 and 10 days of recovery. This was accompanied by an increased inflammatory response in PARP-1 −/− compared with wild-type animals, characterized by neutrophil infiltration and increased IL-6 levels in bronchoalveolar lavages. These lesions were reversible, since the extent of the hyperplastic regions was reduced after 21 days of recovery and did not result in fibrosis. In vitro, lung primary fibroblasts derived from PARP-1 −/− mice showed a higher proliferative response than PARP-1 +/+ cells during air recovery after hyperoxia-induced growth arrest. Altogether, these results reveal an essential role of PARP-1 in the control of cell repair and tissue remodeling after hyperoxia-induced lung injury.
Regulation of cyclooxygenase-2 expression by small GTPase Rac2 in bone marrow macrophages
Authors: Azim Anser C. , Cao Hongmei , Gao Xiaopei , Joo Myungsoo , Malik Asrar B. , van Breemen Richard B. , Sadikot Ruxana T. , Park GyeYoung , Christman John W.
Published: June 2007

Cyclooxygenase 2 (COX-2) is induced by microbial products, proinflammatory cytokines, growth factors, and oncogenes. The Rho family includes RhoA, Rac1, Rac2, Rac3, and Cdc42 and is involved in regulation of the actin cytoskeleton organization, cell growth, vesicular cell trafficking, and transcriptional regulation. Rac2 binds to NADPH oxidase protein complex, and Rac2 null neutrophils are known to have poor phagocytic activity. We examined whether Rac2, the predominant small GTPase in hematopoietic cells, influences COX-2 expression in bone marrow-derived macrophages (BMDM). We showed that BMDM from Rac2−/−null mice have reduced COX-2 expression in response to treatment with endotoxin. Despite a compensatory increase in Rac1, BMDM from Rac2−/−null mice have less biologically active GTP-bound Rac in response to LPS stimulation. Signaling molecules (downstream of Rac2 and Toll-like receptor 4) such as p42/44, p38, and pAKT were also affected in BMDM from Rac2−/−null mouse macrophages. We also observed that BMDM from Rac2−/−null failed to degrade IκBα significantly and had less immunoreactive PU.1. We show that both NF-κB pathway and PU.1 are involved in normal macrophage function and play a role in macrophage COX-2 expression. In summary, these data indicate that Rac2 regulates COX-2 expression in BMDM.
Serotonin transporter protein in pulmonary hypertensive rats treated with atorvastatin
Authors: Laudi Sven , Trump Saskia , Schmitz Volker , West James , McMurtry Ivan F. , Mutlak Haitham , Christians Uwe , Weimann Jörg , Kaisers Udo , Steudel Wolfgang
Published: June 2007

HMG-CoA-reductase inhibitors (statins) influence lipid metabolism and have pleiotropic effects. Several statins reduce various forms of pulmonary hypertension (PH) in animal models. The relationship between atorvastatin and expression of serotonin transporter protein (5-HTT) remains unknown. This study focused on the effects of atorvastatin on the course of monocrotaline (MCT)-induced PH and its relation to 5-HTT expression. Male Sprague-Dawley rats were challenged with MCT with or without subsequent daily oral treatment with 0.1, 1, and 10 mg/kg of atorvastatin for 28 days. Over the 4-wk course, the progression of PH was followed by transthoracic echocardiography [pulmonary artery pressure was assessed by pulmonary artery flow acceleration time (PAAT), an estimate reciprocal to pulmonary artery pressure], and, at the end of the 4-wk course, invasive right ventricular pressure, right ventricular weight, quantitative morphology, and 5-HTT expression were measured. MCT caused significant PH as early as 7 days after injection. Atorvastatin treatment increased PAAT and reduced right ventricular pressure, right ventricular hypertrophy, and vascular remodeling over the 4-wk course. MCT challenge was associated with increased pulmonary vascular 5-HTT expression, and atorvastatin treatment reduced the 5-HTT expression. MCT-induced PH over the course of 4 wk can be easily followed by transthoracic echocardiography, and atorvastatin is effective in reducing the PH. Atorvastatin's effects are associated with a decrease of 5-HTT expression.
The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies
Authors: Carey Michelle A. , Card Jeffrey W. , Voltz James W. , Germolec Dori R. , Korach Kenneth S. , Zeldin Darryl C.
Published: June 2007

Numerous animal studies have revealed significant effects of sex and sex hormones on normal lung development, lung physiology, and various lung diseases. The primary goal of this review is to summarize knowledge to date on the effects of sex and sex hormones on lung development, physiology, and disease in animals. Specific emphasis will be placed on fibrosis, allergic airway disease, acute lung injury models, respiratory infection, and lung toxicology studies.
A novel technique for nonvolitional assessment of quadriceps muscle endurance in humans
Authors: Swallow E. B. , Gosker H. R. , Ward K. A. , Moore A. J. , Dayer M. J. , Hopkinson N. S. , Schols A. M. W. J. , Moxham J. , Polkey M. I.
Published: June 2007

Assessment of quadriceps endurance is of interest to investigators studying human disease. We hypothesized that repetitive magnetic stimulation (rMS) of the intramuscular branches of the femoral nerve could be used to induce and quantify quadriceps endurance. To test this hypothesis, we used a novel stimulating coil to compare the quadriceps endurance properties in eight normal humans and, to confirm that the technique could be used in clinical practice, in eight patients with advanced chronic obstructive pulmonary disease (COPD). To validate the method, we compared in vivo contractile properties of the quadriceps muscle with the fiber-type composition and oxidative enzyme capacity. We used a Magstim Rapid2magnetic nerve stimulator with the coil wrapped around the quadriceps. Stimuli were given at 30 Hz, a duty cycle of 0.4 (2 s on, 3 s off), and for 50 trains. Force generation and the surface electromyogram were measured throughout. Quadriceps twitch force, elicited by supramaximal magnetic stimulation of the femoral nerve, was measured before and after the protocol. Quadriceps muscle biopsies were analyzed for oxidative (citrate synthase, CS) and glycolytic (phosphofructokinase, PFK) enzyme activity and myosin heavy chain isoform protein expression. The time for force to fall to 70% of baseline (T70) was shorter in the COPD group than the control group: 55.6 ± 26.0 vs. 121 ± 38.7 s ( P = 0.0014). Considering patients and controls together, positive correlations were observed between T70and the proportion of type I fibers ( r = 0.68, P = 0.004) and CS-to-PFK ratio (CS/PFK) ( r = 0.67, P = 0.005). We conclude that quadriceps endurance assessed using rMS is feasible in clinical studies.
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

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 >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

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

Antiangiogenic 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

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

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

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

Polymorphonuclear 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]
Regression of Experimental Medulloblastoma following Transfer of HER2-Specific T Cells
Authors: Ahmed Nabil , Ratnayake Maheshika , Savoldo Barbara , Perlaky Laszlo , Dotti Gianpietro , Wels Winfried S. , Bhattacharjee Meenakshi B. , Gilbertson Richard J. , Shine H. David , Weiss Heidi L. , Rooney Cliona M. , Heslop Helen E. , Gottschalk Stephen
Published: June 2007

Medulloblastoma is a common malignant brain tumor of childhood. Human epidermal growth factor receptor 2 (HER2) is expressed by 40% of medulloblastomas and is a risk factor for poor outcome with current aggressive multimodal therapy. In contrast to breast cancer, HER2 is expressed only at low levels in medulloblastomas, rendering monoclonal antibodies ineffective. We determined if T cells grafted with a HER2-specific chimeric antigen receptor (CAR; HER2-specific T cells) recognized and killed HER2-positive medulloblastomas. Ex vivo, stimulation of HER2-specific T cells with HER2-positive medulloblastomas resulted in T-cell proliferation and secretion of IFN-γ and interleukin 2 (IL-2) in a HER2-dependent manner. HER2-specific T cells killed autologous HER2-positive primary medulloblastoma cells and medulloblastoma cell lines in cytotoxicity assays, whereas HER2-negative tumor cells were not killed. No functional difference was observed between HER2-specific T cells generated from medulloblastoma patients and healthy donors. In vivo, the adoptive transfer of HER2-specific T cells resulted in sustained regression of established medulloblastomas in an orthotopic, xenogenic severe combined immunodeficiency model. In contrast, delivery of nontransduced T cells did not change the tumor growth pattern. Adoptive transfer of HER2-specific T cells may represent a promising immunotherapeutic approach for medulloblastoma. [Cancer Res 2007;67(12):5957–64]
Background Mutation Frequency in Microsatellite-Unstable Colorectal Cancer
Authors: Sammalkorpi Heli , Alhopuro Pia , Lehtonen Rainer , Tuimala Jarno , Mecklin Jukka-Pekka , Järvinen Heikki J. , Jiricny Josef , Karhu Auli , Aaltonen Lauri A.
Published: June 2007

Microsatellite instability (MSI) is observed in ∼12% of colorectal cancers. Genes containing a mononucleotide microsatellite in the coding sequence are particularly prone to inactivation in MSI tumorigenesis, and much work has been conducted to identify genes with high repetitive tract mutation rates in these tumors. Much less attention has been paid to background mutation frequencies, and no work has focused on nontranscribed regions. Here, we studied 114 nontranscribed intergenic A/T and C/G repeats 6 to 10 bp in length, located distant from known genes, to examine background mutation frequencies in MSI colorectal cancers. A strong correlation with tract length was observed, and mutation frequencies of up to 87% were observed in 8 to 10 bp tracts. Subsequently, to compare the background mutation rate in transcribed and nontranscribed noncoding repeats, we screened nine randomly selected intronic C/G8 repeats. In addition, the coding repeats of seven suggested MSI target genes, and nine previously published intronic A8 and G8 repeats were analyzed. Intronic repeats seemed to mutate less frequently than nontranscribed intergenic repeats. Our results show that strand slippage mutations in mismatch repair–deficient cells are as abundant in short intergenic repeats as in many proposed MSI target genes. However, under mismatch repair deficiency, strand slippage mutations in transcribed sequences seem to be repaired more efficiently than in intergenic nontranscribed sequences. The mechanisms causing these differences are not yet understood and should be a subject for further studies. For MSI target gene identification, repeats in transcribed sequences seem to be the most appropriate reference group for coding region repeat mutations. [Cancer Res 2007;67(12):5691–8]
Activation of FOXO3a by the Green Tea Polyphenol Epigallocatechin-3-Gallate Induces Estrogen Receptor α Expression Reversing Invasive Phenotype of Breast Cancer Cells
Authors: Belguise Karine , Guo Shangqin , Sonenshein Gail E.
Published: June 2007

Previously, we showed that the bioactive green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibits growth in soft agar of breast cancer cells with Her-2/neu overexpression. Using gene expression profiling, here we show that EGCG treatment of Her-2/neu–driven mammary tumor cells alters the expression of key regulators in the epithelial to mesenchymal transition (EMT) pathway, reducing invasive phenotype. Specifically, the epithelial genes E-cadherin, γ-catenin, MTA3, and estrogen receptor α (ERα) were up-regulated by EGCG, whereas the proinvasive snail gene was down-regulated. Consistently, EGCG inhibited branching colony growth and invasion in Matrigel. EGCG treatment similarly inhibited invasive phenotype of mouse mammary tumor cells driven by Nuclear Factor-κB c-Rel and protein kinase CK2, frequently found overexpressed in human breast disease. Recently, we identified the Forkhead box O transcription factor FOXO3a as a major transcriptional regulator of ERα. Given the pivotal role of ERα in preventing EMT, we hypothesized that the activation of FOXO3a by EGCG plays an important role in the observed reversal of invasive phenotype in ERα-positive breast cancer cells. EGCG treatment activated FOXO3a. Ectopic expression of a constitutively active FOXO3a overrode transforming growth factor-β1–mediated invasive phenotype and induced a more epithelial phenotype, which was dependent on ERα expression and signaling. Conversely, a dominant negative FOXO3a reduced epithelial phenotype of ERα-low breast cancer cells. These results identify, for the first time, a role for FOXO3a in the inhibition of invasive phenotype in breast cancer cells with active ERα signaling and elucidate a novel mechanism whereby EGCG represses EMT of breast cancer cells. [Cancer Res 2007;67(12):5763–70]