bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the Promoter of the Fengycin Synthetase Operon by the UP Element
Authors: Ke Wan-Ju , Chang Ban-Yang , Lin Tsuey-Pin , Liu Shih-Tung
Published: May 2009

Bacillus subtilisF29-3 produces an antifungal peptidic antibiotic that is synthesized nonribosomally by fengycin synthetases. Our previous work established that the promoter of the fengycin synthetase operon is located 86 nucleotides upstream of the translational initiation codon offenC. This investigation involved transcriptional fusions with a DNA fragment that contains the region between positions −105 and +80 and determined that deleting the region between positions −55 and −42 reduces the promoter activity by 64.5%. Transcriptional fusions in theB. subtilisDB2 chromosome also indicated that mutating the sequence markedly reduces the promoter activity. An in vitro transcription analysis confirmed that the transcription is inefficient when the sequence in this region is mutated. Electrophoretic mobility shift and footprinting analyses demonstrated that the C-terminal domain of the RNA polymerase α subunit binds to the region between positions −55 and −39. These results indicated that the sequence is an UP element. Finally, this UP element is critical for the production of fengycin, since mutating the UP sequence in the chromosome ofB. subtilisF29-3 reduces the transcription of thefenoperon by 85% and prevents the cells from producing enough fengycin to suppress the germination ofPaecilomyces variotiispores on agar plates.
The Oligopeptide Transport System Is Essential for the Development of Natural Competence inStreptococcus thermophilusStrain LMD-9
Authors: Gardan Rozenn , Besset Colette , Guillot Alain , Gitton Christophe , Monnet Véronique
Published: May 2009

In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms inStreptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of theS. thermophilusLMD-9 to that of a mutant deleted for the subunits C, D, and E of theamioperon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in theamideletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation inS. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation inS. thermophilusLMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription ofcomX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.
Transcription Activity of IndividualrrnOperons inBacillus subtilisMutants Deficient in (p)ppGpp Synthetase Genes,relA,yjbM, andywaC
Authors: Natori Yousuke , Tagami Kazumi , Murakami Kana , Yoshida Sawako , Tanigawa Osamu , Moh Yoonsuh , Masuda Kenta , Wada Tetsuya , Suzuki Shota , Nanamiya Hideaki , Tozawa Yuzuru , Kawamura Fujio
Published: May 2009

InBacillus subtilisa null mutation of therelAgene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) ofyjbMand/orywaCcoding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, eitheryjbMorywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC inB. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in therelAmutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorlesscatgene. We identified the transcriptional start sites of eachrrnoperon (a G) and found that transcription of allrrnoperons from their P1 promoters was drastically reduced in therelAmutant while this was almost completely restored in therelA yjbM ywaCtriple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in therelAmutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.
Genome Sequencing and Comparative Analysis ofKlebsiella pneumoniaeNTUH-K2044, a Strain Causing Liver Abscess and Meningitis
Authors: Wu Keh-Ming , Li Ling-Hui , Yan Jing-Jou , Tsao Nina , Liao Tsai-Lien , Tsai Hui-Chi , Fung Chang-Phone , Chen Hsiang-Ju , Liu Yen-Ming , Wang Jin-Tung , Fang Chi-Tai , Chang Shan-Chwen , Shu Hung-Yu , Liu Tze-Tze , Chen Ying-Tsong , Shiau Yih-Ru , Lauderdale Tsai-Ling , Su Ih-Jen , Kirby Ralph , Tsai Shih-Feng
Published: May 2009

Nosocomial infections caused by antibiotic-resistantKlebsiella pneumoniaeare emerging as a major health problem worldwide, while community-acquiredK. pneumoniaeinfections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form ofK. pneumoniaethat causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome ofK. pneumoniaeNTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15K. pneumoniaeisolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence ofK. pneumoniaeNTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition amongK. pneumoniaepathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.
Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in
Pseudomonas putida
CBB5
Authors: Yu Chi Li , Louie Tai Man , Summers Ryan , Kale Yogesh , Gopishetty Sridhar , Subramanian Mani
Published: May 2009

Pseudomonas putida
CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.
Complete Genome Sequence ofAggregatibacter(Haemophilus)aphrophilusNJ8700
Authors: Di Bonaventura Maria Pia , DeSalle Rob , Pop Mihai , Nagarajan Niranjan , Figurski David H. , Fine Daniel H. , Kaplan Jeffrey B. , Planet Paul J.
Published: May 2009

We report the finished and annotated genome sequence ofAggregatibacter aphrophilusstrain NJ8700, a strain isolated from the oral flora of a healthy individual, and discuss characteristics that may affect its dual roles in human health and disease. This strain has a rough appearance, and its genome contains genes encoding a type VI secretion system and several factors that may participate in host colonization.
Rapid Identification of Biothreat and Other Clinically Relevant Bacterial Species by Use of Universal PCR Coupled with High-Resolution Melting Analysis
Authors: Yang Samuel , Ramachandran Padmini , Rothman Richard , Hsieh Yu-Hsiang , Hardick Andrew , Won Helen , Kecojevic Aleksandar , Jackman Joany , Gaydos Charlotte
Published: May 2009

A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.
Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41
Authors: Perez Lautaro G. , Costa Matthew R. , Todd Christopher A. , Haynes Barton F. , Montefiori David C.
Published: May 2009

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.
The Paramyxoviruses Simian Virus 5 and Mumps Virus Recruit Host Cell CD46 To Evade Complement-Mediated Neutralization
Authors: Johnson John B. , Grant Ken , Parks Griffith D.
Published: May 2009

The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization.
EnteralClostridium difficile, an emerging cause for high-output ileostomy
Authors: Williams R N , Hemingway D , Miller A S
Published: May 2009

The loss of fluid and electrolytes from a high-output ileostomy (>1200 ml/day) can quickly result in dehydration and if not properly managed may cause acute renal failure. The management of a high-output ileostomy is based upon three principles: correction of electrolyte disturbance and fluid balance, pharmacological reduction of ileostomy output, and treatment of any underlying identifiable cause. There is an increasing body of evidence to suggest thatClostridium difficilemay behave pathologically in the small intestine producing a spectrum of enteritis that mirrors the well-recognised colonic disease manifestation. Clinically this can range from high-output ileostomy to fulminant enteritis. This report describes two cases of high-output ileostomy associated with entericC difficileinfection and proposes that the management algorithm of a high-output ileostomy should include exclusion of small bowelC difficile.
Biotin-free systems provide stronger immunohistochemical signal in oestrogen receptor evaluation of breast cancer
Authors: Rocha R M , Miller K , Soares F , Schenka N , Vassallo J , Gobbi H
Published: May 2009

Aims:

Biotin-free polymeric visualisation systems (BFPS) were compared with streptavidin–biotin systems (SABS) in the evaluation of immunoreactivity for oestrogen receptor (ER) in breast carcinomas.

Methods:

The antiestrogen antibody clone SP1 was employed in a tissue microarray containing 320 breast carcinomas. Eleven different detection systems were used: six second-generation BFPS (Advance, Novolink, SuperPicTure, PicTure Max, Super Sensitive non-biotin HRP and Mouse/Rabbit Polydetector HRP/DAB), one first-generation BFP (EnVision+), and four SABS (LSAB+, EasyPath, Super Sensitive and Mouse/Rabbit Immunodetector HRP/DAB). The slides were digitalised using a Mirax scanner and the resulting images were analysed by an automated method and by visual analysis using the Allred score system considering positive nuclear staining. Cytoplasm staining was also separately evaluated.

Results:

The BFPS Advance and Novolink showed the highest scores by visual analysis, and additionally detected two positive cases that were considered negative using the other detection systems. Likewise, these systems, together with the SAB LSAB+, showed higher staining intensity by the automated method. BFPS revealed no cytoplasm staining, in contrast to the SABS.

Conclusions:

The second-generation BFPS, especially Advance and Novolink, provided stronger and sharper nuclear immunohistochemical signals as compared with most SABS, with no non-specific cytoplasm staining. In a few instances, the second-generation BFPS systems showed discordant results in relation to SABS; therefore further studies correlating these findings to therapeutic responses are necessary. BFPS may represent a high-quality tool for research and clinical evaluation of ER in breast cancer.
Abnormal energetics and ATP depletion in pressure-overload mouse hearts: in vivo high-energy phosphate concentration measures by noninvasive magnetic resonance
Authors: Gupta Ashish , Chacko V. P. , Weiss Robert G.
Published: May 2009

31P magnetic resonance spectroscopy (MRS) offers a unique means to noninvasively quantify the major cardiac high-energy phosphates, creatine phosphate (PCr) and adenosine 5′-triphosphate (ATP), that are critical for normal myocardial contractile function and viability. Spatially localized 31P MRS has been used to quantify the in vivo PCr-to-ATP ratio (PCr/ATP) of murine hearts, including those with pressure-overload hypertrophy induced by thoracic aortic constriction (TAC). To date, there has been no approach for measuring the absolute tissue concentrations of PCr and ATP in the in vivo mouse heart that promise a better understanding of high-energy metabolism. A method to quantify in vivo murine myocardial concentrations of PCr and ATP using an external reference is described, validated, and applied to normal and TAC hearts. This new method does not prolong the scan times in mice beyond those previously required to measure PCr/ATP. The new method renders an [ATP] of 5.0 ± 0.9 (mean ± SD) and [PCr] of 10.4 ± 1.4 μmol/g wet wt in normal mouse hearts ( n = 7) and significantly lower values in TAC hearts ( n = 10) of 4.0 ± 0.8 and 6.7 ± 2.0 μmol/g wet wt for [ATP] ( P < 0.04) and [PCr] ( P < 0.001), respectively. The in vivo magnetic resonance [ATP] results are in good agreement with those obtained using an in vitro enzyme luminescent assay of perchloric acid extracts of the same hearts. In conclusion, a validated 31P MRS method for quantifying [ATP] and [PCr] in the in vivo mouse heart using spatial localization and an external reference is described and used to demonstrate significant reductions in not only PCr/ATP but [ATP] in hypertrophied TAC hearts.
Slow head-up tilt causes lower activation of muscle sympathetic nerve activity: loading speed dependence of orthostatic sympathetic activation in humans
Authors: Kamiya Atsunori , Kawada Toru , Shimizu Shuji , Iwase Satoshi , Sugimachi Masaru , Mano Tadaaki
Published: May 2009

Many earlier human studies have reported that increasing the tilt angle of head-up tilt (HUT) results in greater muscle sympathetic nerve activity (MSNA) response, indicating the amplitude dependence of sympathetic activation in response to orthostatic stress. However, little is known about whether and how the inclining speed of HUT influences the MSNA response to HUT, independent of the magnitude of HUT. Twelve healthy subjects participated in passive 30° HUT tests at inclining speeds of 1° (control), 0.1° (slow), and 0.0167° (very slow) per second. We recorded MSNA (tibial nerve) by microneurography and assessed nonstationary time-dependent changes of R-R interval variability using a complex demodulation technique. MSNA averaged over every 10° tilt angle increased during inclination from 0° to 30°, with smaller increases in the slow and very slow tests than in the control test. Although a 3-min MSNA overshoot after reaching 30° HUT was observed in the control test, no overshoot was detected in the slow and very slow tests. In contrast with MSNA, increases in heart rate during the inclination and after reaching 30° were similar in these tests, probably because when compared with the control test, greater increases in plasma epinephrine counteracted smaller autonomic responses in the very slow test. These results indicate that slower HUT results in lower activation of MSNA, suggesting that HUT-induced sympathetic activation depends partially on the speed of inclination during HUT in humans.
Myocardial TLR4 is a determinant of neutrophil infiltration after global myocardial ischemia: mediating KC and MCP-1 expression induced by extracellular HSC70
Authors: Ao Lihua , Zou Ning , Cleveland Joseph C. , Fullerton David A. , Meng Xianzhong
Published: May 2009

Cardiac surgery with global myocardial ischemia-reperfusion (I/R) induces a myocardial inflammatory response that impairs cardiac recovery. Chemokines contribute to the overall myocardial inflammatory response through inducing leukocyte infiltration. Although Toll-like receptor 4 (TLR4) has an important role in postischemic myocardial injury, the relative roles of myocardial tissue and leukocyte TLR4 in leukocyte infiltration, as well as the role of TLR4 in myocardial chemokine expression, are unclear. Our recent study, in an isolated mouse heart model of global I/R, found that the 70-kDa heat shock cognate protein (HSC70) is released from cardiac cells and mediates the expression of cardiodepressant cytokines via a TLR4-dependent mechanism. In the present study, we tested the hypotheses that myocardial tissue TLR4 has a major role in mediating neutrophil infiltration and that myocardial TLR4 and extracellular HSC70 contribute to the mechanisms underlying cardiac chemokine response to global I/R. We subjected hearts isolated from TLR4-defective and TLR4-competent mice to global I/R and examined myocardial neutrophil infiltration and expression of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1 (MCP-1). TLR4-defective hearts exhibited reduced neutrophil infiltration regardless of the phenotypes of neutrophils perfused during reperfusion and expressed lower levels of KC and MCP-1. HSC70-specific antibody reduced myocardial expression of KC and MCP-1 after I/R. Furthermore, perfusion of HSC70 increased KC and MCP-1 expression in TLR4-competent hearts but not in TLR4-defective hearts, and HSC70 also induced the chemokine response in macrophages in a TLR4-dependent fashion. A recombinant HSC70 fragment lacking the substrate-binding domain was insufficient to induce chemokine expression in hearts and cells. This study demonstrates that myocardial tissue TLR4, rather than neutrophil TLR4, is the determinant of myocardial neutrophil infiltration after global I/R. TLR4 mediates myocardial chemokine expression, and the mechanisms involve extracellular HSC70. These results imply the HSC70-TLR4 interaction as a novel mechanism underlying the myocardial chemokine response to global I/R.
Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice
Authors: Barrick Cordelia J. , Roberts Reade B. , Rojas Mauricio , Rajamannan Nalini M. , Suitt Carolyn B. , O'Brien Kevin D. , Smyth Susan S. , Threadgill David W.
Published: May 2009

Epidermal growth factor receptor (EGFR) signaling contributes to aortic valve development in mice. Because developmental phenotypes in Egfr-null mice are dependent on genetic background, the hypomorphic Egfr wa2 allele was made congenic on C57BL/6J (B6) and 129S1/SvImJ (129) backgrounds and used to identify the underlying cellular cause of EGFR-related aortic valve abnormalities. Egfr wa2/wa2 mice on both genetic backgrounds develop aortic valve hyperplasia. Many B6- Egfr wa2/wa2 mice die before weaning, and those surviving to 3 mo of age or older develop severe left ventricular hypertrophy and heart failure. The cardiac phenotype was accompanied by significantly thicker aortic cusps and larger transvalvular gradients in B6- Egfr wa2/wa2 mice compared with heterozygous controls and age-matched Egfr wa2 homozygous mice on either 129 or B6129F1 backgrounds. Histological analysis revealed cellular changes in B6- Egfr wa2/wa2 aortic valves underlying elevated pressure gradients and progression to heart failure, including increased cellular proliferation, ectopic cartilage formation, extensive calcification, and inflammatory infiltrate, mimicking changes seen in human calcific aortic stenosis. Despite having congenitally enlarged valves, 129 and B6129F1- Egfr wa2/wa2 mice have normal lifespans, absence of left ventricular hypertrophy, and normal systolic function. These results show the requirement of EGFR activity for normal valvulogenesis and demonstrate that dominantly acting genetic modifiers curtail pathological changes in congenitally deformed valves. These studies provide a novel model of aortic sclerosis and stenosis and suggest that long-term inhibition of EGFR signaling for cancer therapy may have unexpected consequences on aortic valves in susceptible individuals.
Insulin receptor substrate-1/2 mediates IL-4-induced migration of human airway epithelial cells
Authors: White Steven R. , Martin Linda D. , Abe Mark K. , Marroquin Bertha A. , Stern Randi , Fu Xiaoying
Published: May 2009

Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-1 or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2.
GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP
Authors: Kumar Sanjiv , Sun Xutong , Sharma Shruti , Aggarwal Saurabh , Ravi Kandasamy , Fineman Jeffery R. , Black Stephen M.
Published: May 2009

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH4) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH4could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH4levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH4levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH4levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH4levels.
Atropine-enhanced, antigen challenge-induced airway hyperreactivity in guinea pigs is mediated by eosinophils and nerve growth factor
Authors: Verbout Norah G. , Jacoby David B. , Gleich Gerald J. , Fryer Allison D.
Published: May 2009

Although anticholinergic therapy inhibits bronchoconstriction in asthmatic patients and antigen-challenged animals, administration of atropine 1 h before antigen challenge significantly potentiates airway hyperreactivity and eosinophil activation measured 24 h later. This potentiation in airway hyperreactivity is related to increased eosinophil activation and is mediated at the level of the airway nerves. Since eosinophils produce nerve growth factor (NGF), which is known to play a role in antigen-induced airway hyperreactivity, we tested whether NGF mediates atropine-enhanced, antigen challenge-induced hyperreactivity. Antibody to NGF (Ab NGF) was administered to sensitized guinea pigs with and without atropine pretreatment (1 mg/kg iv) 1 h before challenge. At 24 h after challenge, animals were anesthetized, vagotomized, paralyzed, and ventilated. Electrical stimulation of both vagus nerves caused bronchoconstriction that was increased in challenged animals. Atropine pretreatment potentiated antigen challenge-induced hyperreactivity. Ab NGF did not affect eosinophils or inflammatory cells in any group, nor did it prevent hyperreactivity in challenged animals that were not pretreated with atropine. However, Ab NGF did prevent atropine-enhanced, antigen challenge-induced hyperreactivity and eosinophil activation (assessed by immunohistochemistry). This effect was specific to NGF, since animals given control IgG remained hyperreactive. These data suggest that anticholinergic therapy amplifies eosinophil interactions with airway nerves via NGF. Therefore, therapeutic strategies that target both eosinophil activation and NGF-mediated inflammatory processes in allergic asthma are likely to be beneficial.
Resolution of renal inflammation: a new role for NF-κB1 (p50) in inflammatory kidney diseases
Authors: Panzer Ulf , Steinmetz Oliver M. , Turner Jan-Eric , Meyer-Schwesinger Catherine , von Ruffer Claudia , Meyer Tobias N. , Zahner Gunther , Gómez-Guerrero Carmen , Schmid Roland M. , Helmchen Udo , Moeckel Gilbert W. , Wolf Gunter , Stahl Rolf A. K. , Thaiss Friedrich
Published: May 2009

In renal tissue injury, activation of the transcription factor NF-κB has a central role in the induction of proinflammatory gene expression, which are involved in the development of progressive renal inflammatory disease. The function of NF-κB during the switch from the inflammatory process toward resolution, however, is largely unknown. Therefore, we assessed the time-dependent activation and function of NF-κB in two different models of acute nephritis. Our experiments demonstrate a biphasic activation of NF-κB in the anti-Thy-1 model of glomerulonephritis in rats and the LPS-induced nephritis in mice, with a first peak during the induction phase and a second peak during the resolution period. After induction of glomerular immune injury in rats, predominantly NF-κB p65/p50 heterodimer complexes are shifted to the nucleus whereas during the resolution phase predominantly p50 homodimers could be demonstrated in the nuclear compartment. In addition, we could demonstrate that p50 protein plays a pivotal role in the resolution of LPS-induced renal inflammation since NF-κB p50 knockout mice demonstrate significantly higher chemokine expression, prolonged renal inflammatory cell infiltration with consecutive tissue injury, and reduced survival. In conclusion, our studies indicate that NF-κB subunit p50 proteins have critical in vivo functions in immunologically mediated renal disease by downregulating inflammation during the resolution period.
Castration-Resistant Prostate Cancer: Locking Up the Molecular Escape Routes
Authors: Attar Ricardo M. , Takimoto Chris H. , Gottardis Marco M.
Published: May 2009

The understanding of the key role that androgens play on the normal and pathological physiology of the prostate guided the development of different therapies for the treatment of locally advanced or metastatic prostate cancer (PCa). These so-called androgen deprivation therapies include surgical or chemical castration, achieved by the administration of gonadotropin-releasing hormone analogs; inhibition of steroidogenic enzymes; and finally, blocking of the binding of androgens to their receptor (AR) by the use of antiandrogens. Despite an excellent initial response, in approximately 2 to 3 years, most of these patients will succumb to the castration resistant form of the disease. Remarkably, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional AR, and several molecular mechanisms have been proposed to explain this phenomenon. These include: (1) gene amplification and increased expression of the AR mRNA and protein, (2) selection of mutations in the AR that confer broader ligand specificity, (3) changes in the ratios or expression between the AR and its coregulators, (4) increased expression of steroidogenic enzymes, and (5) up-regulation of cross-talk signal transduction pathways that can activate the AR in a ligand-independent manner. We will summarize how these molecular hypotheses are being tested in the clinic by the latest therapeutic modalities.
Nuclear Factor-κB-Mediated Transforming Growth Factor-β-Induced Expression of Vimentin Is an Independent Predictor of Biochemical Recurrence after Radical Prostatectomy
Authors: Zhang Qiang , Helfand Brian T. , Jang Thomas L. , Zhu Lihua J. , Chen Lin , Yang Ximing J. , Kozlowski James , Smith Norm , Kundu Shilajit D. , Yang Guangyu , Raji Adekunle A. , Javonovic Borko , Pins Michael , Lindholm Paul , Guo Yinglu , Catalona William J. , Lee Chung
Published: May 2009

Purpose: Transforming growth factor-β (TGF-β)-mediated epithelial-to-mesenchymal transition (EMT) has been shown to occur in some cancers; however, the pathway remains controversial and varies with different cancers. In addition, the mechanisms by which TGF-β and the EMT contribute to prostate cancer recurrence are largely unknown. In this study, we elucidated TGF-β-mediated EMT as a predictor of disease recurrence after therapy for prostate cancer, which has not been reported before.

Experimental Design: We analyzed TGF-β-induced EMT using nuclear factor-κB (NF-κB) as an intermediate mediator in prostate cancer cell lines. A total of 287 radical prostatectomy specimens were evaluated using immunohistochemistry in a high-throughput tissue microarray analysis. Levels of TGF-β signaling components and EMT-related factors were analyzed using specific antibodies. Results were expressed as the percentage of cancer cells that stained positive for a given antibody and were correlated with disease recurrence rates at a mean of 7 years following radical prostatectomy.

Results: In prostate cancer cell lines, TGF-β-induced EMT was mediated by NF-κB signaling. Blockade of NF-κB or TGF-β signaling resulted in abrogation of vimentin expression and inhibition of the invasive capability of these cells. There was high risk of biochemical recurrence associated with tumors that displayed high levels of expression of TGF-β1, vimentin, and NF-κB and low level of cytokeratin 18. This was particularly true for vimentin, which is independent of patients' Gleason score.

Conclusions: The detection of NF-κB-mediated TGF-β-induced EMT in primary tumors predicts disease recurrence in prostate cancer patients following radical prostatectomy. The changes in TGF-β signaling and EMT-related factors provide novel molecular markers that may predict prostate cancer outcomes following treatment.
Inhibition of Src Phosphorylation Alters Metastatic Potential of Osteosarcoma In vitro but not In vivo
Authors: Hingorani Pooja , Zhang Wendong , Gorlick Richard , Kolb E. Anders
Published: May 2009

Purpose: Pulmonary metastasis remains the major cause of mortality in osteosarcoma. Src tyrosine kinase is a key player involved in metastatic pathways in multiple human cancers. c-Src has been shown to be expressed and phosphorylated in osteosarcoma cell lines and inhibiting Src phosphorylation in these cells causes inhibition of the metastatic phenotype in vitro. We studied the effect of inhibition of Src phosphorylation in preventing the growth and development of pulmonary metastases in osteosarcoma.

Experimental Design: Dasatinib, a dual Src-Abl kinase inhibitor, was used to study the effect of Src kinase inhibition on proliferation, adhesion, and invasion of osteosarcoma cell lines in vitro and in preventing the development of pulmonary metastases in a spontaneously metastatic mouse model.

Results: In vitro, phosphorylation of Src and its downstream signaling molecules such as focal adhesion kinase, Crk-associated substrate, and c-Jun was inhibited at nanomolar concentrations of dasatinib. Dasatinib was not cytotoxic against the osteosarcoma cells with the IC50 ranging from 10 to 20 μmol/L but effectively inhibited the adhesion and migration of osteosarcoma cells at 10 to 100 nmol/L. However, in vivo, dasatinib did not inhibit the development of pulmonary metastases despite complete inhibition of Src phosphorylation in the primary tumors. No effect was seen in the primary tumor growth and the degree of apoptosis.

Conclusions: These results suggest that Src kinase activation might not be the primary pathway involved in the development of pulmonary metastases in osteosarcoma.
Src Family Kinase Activity Is Up-Regulated in Hormone-Refractory Prostate Cancer
Authors: Tatarov Oleg , Mitchell Thomas J. , Seywright Morag , Leung Hing Y. , Brunton Valerie G. , Edwards Joanne
Published: May 2009

Purpose: Although Src family kinase (SFK) inhibitors are now in clinical trials for the treatment of androgen-independent prostate cancer (AIPC), there are no studies relating SFK activation to patient survival. This study was designed to determine if SFK activation was up-regulated with the development of AIPC and if patients could be selected who were more likely to respond to therapy.

Experimental Design: A unique cohort of matched prostate tumor samples, taken before hormone deprivation therapy and following hormone relapse, was used to determine by immunohistochemistry on an individual patient basis if SFK activity changed with progression to AIPC and whether this related to patient outcome measures. Using matched, hormone-sensitive and hormone-refractory cell lines, we determined if hormone status affected the way prostate cancer cells respond to suppression of SFK activity by the small-molecule inhibitor dasatinib.

Results: In the current study, 28% of patients with AIPC exhibited an increase in SFK activity in prostate cancer tissue, these patients had significantly shorter overall survival (P<0.0001), and activated SFK expression correlated with the presence of distant metastases. Dasatinib inhibited phosphorylation of Src and Lyn and the downstream substrate FAK in hormone-sensitive and hormone-refractory cell lines. Although migration was reduced by dasatinib in both cell lines, proliferation of hormone-refractory cells only was inhibited.

Conclusion: Appropriate patient selection may allow better targeting of prostate cancer patients who are likely to respond to the treatment with SFK inhibitors at the same time improving the outcome of clinical trials.
Aberrant Splicing ofHugl-1Is Associated with Hepatocellular Carcinoma Progression
Authors: Lu Xuefeng , Feng Xiujing , Man Xiaobo , Yang Guang , Tang Liang , Du Dan , Zhang Fan , Yuan Haixin , Huang Qin , Zhang Zhe , Liu Yinkun , Strand Dennis , Chen Zhengjun
Published: May 2009

Purpose: Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC).

Experimental Design: Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay.

Results: In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short “direct repeat” sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice.

Conclusions: We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.
An Akt/Hypoxia-Inducible Factor-1α/Platelet-Derived Growth Factor-BB Autocrine Loop Mediates Hypoxia-Induced Chemoresistance in Liver Cancer Cells and Tumorigenic Hepatic Progenitor Cells
Authors: Lau Chi Keung , Yang Zhen Fan , Ho David W. , Ng Michael N. , Yeoh George C.T. , Poon Ronnie T.P. , Fan Sheung Tat
Published: May 2009

Purpose: The goals of the present study were to investigate the mechanism of hypoxia-mediated chemoresistance in liver cancer cells and tumorigenic hepatic progenitor (oval) cells and to determine whether disrupting an Akt/hypoxia-inducible factor-1α (HIF-1α)/platelet-derived growth factor (PDGF)-BB autocrine loop can enhance chemotherapeutic efficacy in hypoxia.

Experimental Design: Five hepatocellular carcinoma (HCC) cell lines and two hepatic progenitor cell lines were treated in vitro with cisplatin under both normoxic and hypoxic conditions. To generate ischemic hypoxia for tumor cells in vivo, hepatic artery ligation was applied to an orthotopic HCC model. Cisplatin and YC1, which is a HIF-1α inhibitor, were administered by portal vein and intratumoral injections, respectively.

Results: Cell viability was higher under hypoxic than normoxic conditions. HIF-1α and Akt were up-regulated under hypoxic conditions, forming an autocrine signaling loop with PDGF-BB. Akt/HIF-1α/PDGF-BB signaling regulated Akt to confer cisplatin resistance to HCC cell lines in vitro. This autocrine signaling loop also contributed to chemoresistance in the tumorigenic hepatic progenitor cell line PIL2 under hypoxic conditions but not in the nontumorigenic cell line PIL4. In an orthotopic HCC model, combining blockade of HIF-1α activity with ischemic hypoxia significantly enhanced the efficacy of chemotherapy, leading to suppression of tumor growth and prolongation of animal survival.

Conclusion: Blockade of Akt/HIF-1α/PDGF-BB autocrine signaling could enhance the chemosensitivity of liver cancer cells and tumorigenic hepatic progenitor cells under hypoxic conditions and thus provide an effective therapeutic strategy for HCC.
Immunodeficient Mouse Strains Display Marked Variability in Growth of Human Melanoma Lung Metastases
Authors: Carreno Beatriz M. , Garbow Joel R. , Kolar Grant R. , Jackson Erin N. , Engelbach John A. , Becker-Hapak Michelle , Carayannopoulos Leonidas N. , Piwnica-Worms David , Linette Gerald P.
Published: May 2009

Purpose: Immunodeficient mice serve as critical hosts for transplantation of xenogeneic cells for in vivo analysis of various biological processes. Because investigators typically select one or two immunodeficient mouse strains as recipients, no comprehensive study has been published documenting differences in human tumor engraftment. Taking advantage of the increased metastatic potential of RhoC-expressing human (A375) melanoma cells, we evaluate four immunodeficient mouse strains: severe combined immunodeficiency (scid), nonobese diabetic (NOD)-scid, NOD-scid β2mnull, and NOD-scid IL2Rγnull as xenograft tumor recipients.

Experimental Design: Bioluminescence, magnetic resonance imaging, and histopathology were used to monitor serial tumor growth. Natural killer (NK) cell function was examined in each mouse strain using standard 51Chromium release assays.

Results: Melanoma metastases growth is delayed and variable in scid and NOD-scid mice. In contrast, NOD-scid β2mnull and NOD-scid IL2Rγnull mice show rapid tumor engraftment, although tumor growth is variable in NOD-scid β2mnull mice. NK cells were detected in all strains except NOD-scid IL2Rγnull, and in vitro activated scid, NOD-scid, and NOD-scid β2mnull NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands MHC class I chain–related A and B molecules renders melanoma susceptible to murine NK cell–mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D.

Conclusions: Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction used by NK cells in immunodeficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rγnull animals makes this strain an excellent recipient of melanoma and potentially other human malignancies.